Dna fragment carrying toluene monooxygenase gene, recombinant plasmid, transformed microorganism, method for degrading chlorinated aliphatic hydrocarbon compounds and aromatic compounds, and method for environmental remediation

ABSTRACT

A recombinant DNA is constructed by using a toluene monooxygenase gene isolated from  Burkholderia cepacia  strain KK01 and employed to provide the transformant which can express toluene monooxygenase useful for cleaning of aqueous media such as drain and waste water containing chlorinated aliphatic hydrocarbon compounds or aromatic compounds, for remediation of soil polluted with such compounds, and cleaning of air (gas phase) polluted with volatile organic chlorine compounds.

BACKGROUND OF THE INVENTION

[0001] 1. Field of the Invention

[0002] The present invention relates to a novel DNA fragment carrying atoluene monooxygenase gene, a novel recombinant DNA containing the DNAfragment, a transformant containing the recombinant DNA, and a methodfor degrading chlorinated aliphatic hydrocarbon compounds such astrichloroethylene (TCE) and dichloroethylene (DCE) and aromaticcompounds such as toluene, benzene, phenol, and cresol. The presentinvention also relates to a method for environmental remediation usefulfor cleaning of aqueous media such as wastewater and effluent containingat least either a chlorinated aliphatic hydrocarbon compound or anaromatic compound and air (gas phase) and soil polluted with chlorinatedaliphatic hydrocarbon compounds.

[0003] 2. Related Background Art

[0004] Recently, it has become a serious problem the environmentalpollution with volatile organic chlorinated compounds which are harmfulto the organisms and hardly degradable. Especially, the soil in theindustrial areas in Japan as well as abroad is considered to becontaminated with chlorinated aliphatic hydrocarbon compounds such astetrachloroethylene (PCE), trichloroethylene (TCE), and dichloroethylene(DCE) and aromatic compounds such as toluene, benzene, phenol, andcresol. There have been a number of reports on actual detection of suchpollutants through environmental surveys. It is supposed that thesecompounds remaining in soil dissolve in ground water via rainwater, andthereby spread over the surrounding areas. There is a strong suspicionthat these compounds are carcinogens, and further, these are quitestable in the environment; therefore contamination of groundwater, whichis used as a source of drinking water, has become a serious socialproblem. Therefore, cleaning of aqueous media such as contaminatedgroundwater and soil through removal and degradation of these compoundsand accompanying cleaning of the surrounding gas phase are quiteimportant in view of the environment protection. Technologies requiredfor cleaning (for example, adsorption treatment using activated carbon,degradation treatment using light and heat) have been developed.Technologies presently available, however, are not always practical interms of cost and operability. Recently, microbial degradation ofchlorinated aliphatic hydrocarbon compounds such as TCE that is stablein environment has been reported. The microbial degradation method haveadvantages such as: (1) degradation of chlorinated aliphatic hydrocarboncompounds into harmless substances by using appropriately selectedmicroorganism; (2) no requirement for any special chemicals inprinciple; and (3) reduction of the labor and costs of maintenance.

[0005] The examples of microorganisms capable of degrading TCE are asfollows:

[0006]Welchia alkenophila sero 5 (U.S. Pat. No. 4877736, ATCC 53570,Welchia alkenophila sero 33 (U.S. Pat. No. 4877736, ATCC 53571),Methylocystis sp. Strain M (Agric. Biol. Chem., 53, 2903 (1989), Biosci.Biotech. Bichem., 56, 486 (1992), ibid. 56, 736 (1992)), Methylosinustrichosporium OB3b (Am. Chem. Soc. Natl. meet. Div. Environ. Microbiol.,29, 365 (1989), Appl. Environ. Microbiol., 55, 3155 (1989), Appl.Biochem. Biotechnol. 28, 877 (1991), Japanese Patent ApplicationLaid-Open No. 2-92274 specification, Japanese Patent Laid-OpenApplication No. 3-292970), Methylomonas sp. MM2 (Appl. Environ.Microbiol., 57, 236 (1991), Alcaligenes denitrificans ssp. XylosoxidansJE75 (Arch. Microbiol., 154, 410 (1990), Alcaligenes eutrophus JMP134(Appl. Environ. Microbiol., 56, 1179 (1990), Alcaliqenes eutrophusFERM-13761 (Japanese Patent Laid-Open Application No. 7-123976),Pseudomonas aeruginosa J1104 (Japanese Patent Application Laid-Open No.7-236895), Mycobacterium vaccae JOB5 (J. Gen. Microbiol., 82, 163(1974), Appl. Environ. Microbiol., 55, 2960 (1989), ATCC 29678),Pseudomonas putida BH (Gesuidou Kyoukai-shi (Japan Sewage WorksAssociation Journal), 24, 27 (1987)), Pseudomonas sp. strain G4 (Appl.Environ. Microbiol., 52, 383 (1968), ibid. 53, 949 (1987), ibid. 54, 951(1988), ibid. 56, 279 (1990), ibid. 57, 193 (1991), U.S. Pat. No.4925802, ATCC 53617, this strain was first classified as Pseudomonascepacia and then changed to Pseudomonas sp.), Pseudomonas mendocia KR-1(Bio/Technol., 7, 282 (1989)), Pseudomonas putida F1 (Appl. EnvironMicrobiol., 54, 1703 (1988), ibid. 54, 2578 (1988)), Pseudomonasfluorescens PFL12 (Appl. Environ. Microbiol., 54, 2578 (1988)),Pseudomonas putida KWI-9 (Japanese Patent Application Laid-Open No.6-70753), Pseudomonas cepacia KK01 (Japanese Patent ApplicationLaid-Open No. 6-22769 ), Nitrosomonas europaea (Appl. Environ.Microbio., 56, 1169 (1990), Lactobacillus vaginalis sp. nov (Int. J.Syst. Bacteriol., 39, 368 (1989), ATCC 49540), Nocardia corallina B-276(Japanese Patent Application Laid-Open No. 8-70881, FERM BP-5124, ATCC31338), and so on.

[0007] The problem in actually using these degrading microorganisms inenvironmental remediation treatment, however, resides in optimizing andmaintaining expression of their degradation activity for chlorinatedaliphatic hydrocarbon compounds such as TCE. In an environmentalremediation treatment which utilizes phenol, toluene, methane, or thelike as an inducer, continuous supply of the inducer is indispensable,since depletion of such inducers directly results in stoppage ofdegradation of chlorinated aliphatic hydrocarbon compounds. Presence ofsuch inducers, on the other hand, may inhibit the efficient degradationof the target substance such as TCE, since the affinity of thechlorinated aliphatic hydrocarbon compounds such as TCE as a substrateis considerably low in comparison with these inducers. In addition,precise control of the inducer concentration on the treatment spot isdifficult.

[0008] Thus, use of an inducer is a large problem in practicalapplication of environmental remediation treatment utilizingmicroorganisms.

[0009] In order to solve the problem, Nelson et al. developed a methodusing tryptophan as an inducer for degradation of volatile organicchlorinated compounds (Japanese Patent Application Laid-Open No.4-502277). Tryptophan, however, is a very expensive substance, andalthough tryptophane has no toxicity or risk as a substance, it is notpreferable to introduce excessive carbon and nitrogen sources intoenvironment since it may induce eutrophication. In addition, the problemthat tryptophan serves as a competitive inhibitor in degradation of TCEstill remains.

[0010] Shields et al. obtained a mutant strain of Pseudomonas cepacia G4(changed to Pseudomonas sp. upon deposition to ATCC) by the transposontechnique, which mutant strain does not require an inducer (in thiscase, phenol or toluene) and can degrade TCE (Appl. Environ. Microbiol.,58, 3977 (1992), International Publication No. WO/19738). Also, a mutantnot requiring methane as the inducer has been isolated from Methylosinustrichosporium OB3b, a methanotroph capable of degrading TCE (U.S. Pat.No. 5316940).

[0011] Japanese Patent Application Laid-Open No. 8-294387 also disclosesstrain JM1 (FERM BP-5352) capable of degrading volatile organicchlorinated compounds and aromatic compounds without requiring aninducer, isolated by nitrosoguanidine mutagenization of strain J1 (FERMBP-5102). While, it has been studied to introduce resting cellsexpressing TCE-degrading activity into the remediation site after thepreculture of the cells in the presence of an inducer (Environ. Sci.Technol., 30, 1982 (1996)).

[0012] It has been reported that remediation treatment not requiring theinducer actually makes the remediation treatment easy and efficientcompared to the conventional treatment using inducers.

[0013] However, the growth control of the degrading microorganisms isvery important for both the expression of the degradation activity ondemand and the continuation of degradation. When resting cells are used,it is a problem to be solved that TCE cannot be degraded beyond theamount and period of degradation capacity of the introduced restingcells. In addition, in a large scale treatment, there are furtherproblems that degradation activity will decrease since it takes a longtime to prepare resting cells; the treating apparatus must be large inscale; treatment process is complicated; and the cost may be unfavorablyhigh. Accordingly, it has been attempted to introduce a plasmid carryinga DNA fragment containing a gene region encoding oxygenase orhydroxylase into a host microorganism to make the host express the TCEdegradation activity constitutively or inducibly using a harmlessinducer. For example, there are Pseudomonas mendocina KR-1 (JapanesePatent Application Laid-Open No. 2-503866, Pseudomonas putida KWI-9(Japanese Patent Application Laid-Open No. 6-105691), Pseudomonas putidaBH (Summary of 3rd Conference on Pollution of Ground Water/Soil and ItsProtective Countermeasure, p.213 (1994)), and a transformant carryingboth a toluene degradation enzyme gene derived from Pseudomonas putidaF1 and a biphenyl degradation enzyme gene derived from Pseudomonaspseudoalkaligenes (Japanese Patent Application Laid-Open No. 7-143882).However, the reported TCE degradation activity of the transformants arelow, and the advantages of the transformants has not been fully utilizedfor efficient degradation of TCE, such as the ease of degradationcontrol, freedom in designing recombinant, and no requirements forinducers.

SUMMARY OF THE INVENTION

[0014] It is an object of the present invention to provide a novel DNAfragment encoding a toluene monooxygenase of a high efficiency indegrading aromatic compounds and/or organic chorine compounds, a novelrecombinant DNA containing the DNA fragment, and a transformantcontaining the recombinant DNA. It is another object of the presentinvention to provide an efficient biodegradation method for volatileorganic chlorinated compounds such as trichloroethylene (TCE) anddichloroethylene (DCE) and aromatic compounds such as toluene, benzene,phenol, and cresol using the transformant, specifically an efficientenvironmental remediation method useful for purifying aqueous media suchas wastewater and effluent containing chlorinated aliphatic hydrocarboncompounds or aromatic compounds, remedying soil polluted withchlorinated aliphatic hydrocarbon compounds or aromatic compounds, andpurifying air (gas phase) polluted with chlorinated aliphatichydrocarbon compounds.

[0015] To achieve the above objects, the inventors of the presentinvention strained to isolate the gene encoding toluene monooxygenasefrom Burkholderia cepacia KK01 (previously Pseudomonas cepacia,deposited in the National Institute of Bioscience and Human Technology,Agency of Industrial Science and Technology in accordance with therequirements of the Budapest Treaty, Deposit Date: Mar. 11, 1992,Accession No. FERM BP-4235) having a toluene monooxygenase that oxidizestoluene to ortho-cresol and 3-methylcatechol. Successful isolation andcharacterization of the gene completed the present invention.

[0016] According to one aspect of the present invention, there isprovided a DNA fragment of about 5.8 Kb containing a toluenemonooxygenase gene having a following restriction map, where 1 BamHIrestriction site, 2 EcoRI restriction sites, 1 HpaI restriction site, 1KpnI restriction site, 1 NcoI restriction site, 1 NspV restriction site,1 SacI restriction site, 2 SmaI restriction sites, 3 SphI restrictionsites, 2 XhoI restriction sites, no ClaI restriction site, no DraIrestriction site, no EcoRV restriction site, no HindIII restrictionsite, no NdeI restriction site, no NheI restriction site, no PvuIIrestriction site, no ScaI restriction site, no Sse8387I restrictionsite, no StuI restriction site, and no XbaI restriction site arepresent.

[0017] According to another embodiment of the present invention, thereis provided a DNA fragment having the nucleotide sequence of SEQ ID NO:1with deletion, substitution and/or addition of one or more nucleotides,still encoding an active toluene monooxygenase.

[0018] Further, according to one aspect of the present invention, thereis provided a recombinant DNA comprising a vector enabling maintenanceor replication in a host and a DNA fragment of about 5.8 Kb containing atoluene monooxygenase gene having a following restriction map, where 1BamHI restriction site, 2 EcoRI restriction sites, 1 HpaI restrictionsite, 1 KpnI restriction site, 1 NcoI restriction site, 1 NspVrestriction site, 1 SacI restriction site, 2 SmaI restriction sites, 3SphI restriction sites, 2 XhoI restriction sites, no ClaI restrictionsite, no DraI restriction site, no EcoRV restriction site, no HindIIIrestriction site, no NdeI restriction site, no NheI restriction site, noPvuII restriction site, no ScaI restriction site, no Sse8387Irestriction site, no StuI restriction site, and no XbaI restriction siteare present.

[0019] Further, according to another embodiment of the presentinvention, there is provided another recombinant DNA comprising a vectorenabling maintenance or replication in a host, and a DNA fragmentligated thereto having the nucleotide sequence of SEQ ID NO:1 withdeletion, substitution and/or addition of one or more bases, stillencoding an active toluene monooxygenase.

[0020] According to still another aspect of the present invention, thereis provided another recombinant DNA comprising a vector enablingmaintenance or replication in a host, and a DNA fragment containing aregion encoding a toluene monooxygenase, where the region comprises afirst sequence encoding a polypeptide TomL having an amino acid sequenceof SEQ ID NO:3, a second sequence encoding a polypeptide TomM having anamino acid sequence of SEQ ID NO:4, a third sequence encoding apolypeptide TomN having an amino acid sequence of SEQ ID NO:5, a fourthsequence encoding a polypeptide TomO having an amino acid sequence ofSEQ ID NO:6, and a fifth sequence encoding a polypeptide TomP having anamino acid sequence of SEQ ID NO:7, and the first to fifth sequences arealigned so that expressed TomL-TomP can form an active monooxygenaseprotein.

[0021] According to still another aspect of the present invention, thereis provided another recombinant DNA comprising a vector enablingmaintenance or replication in a host, and a DNA fragment containing aregion encoding a toluene monooxygenase, where the region comprises afirst sequence encoding a polypeptide TomL having an amino acid sequenceof SEQ ID NO:3, a second sequence encoding a polypeptide TomM having anamino acid sequence of SEQ ID NO:4, a third sequence encoding apolypeptide TomN having an amino acid sequence of SEQ ID NO:5, a fourthsequence encoding a polypeptide TomO having an amino acid sequence ofSEQ ID NO:6, and a fifth sequence encoding a polypeptide TomP having anamino acid sequence of SEQ ID NO:7, and the first to fifth sequences arealigned so that expressed TomL-TomP can form an active monooxygenaseprotein, wherein one or more nucleotides have been deleted, substituted,or added in at least one of the sequences with the proviso that theactivity of toluene monooxygenase is not impaired.

[0022] According to still another aspect of the present invention, thereis provided a DNA fragment containing a region encoding a polypeptideTomK having an amino acid sequence of SEQ ID NO:2 wherein the functionof TomK is to enhance a toluene monooxygenase activity of a proteinconsisting at least of TomL to TomP, or encoding a variant TomK havingan amino acid sequence varied from SEQ ID NO:2 with the proviso that thefunction of TomK is not impaired.

[0023] According to still another aspect of the present invention, thereis provided a recombinant DNA comprising a vector; a promoter; and a DNAfragment containing a region encoding a toluene monooxygenase, where theregion comprises a first sequence encoding a polypeptide TomL having anamino acid sequence of SEQ ID NO:3, a second sequence encoding apolypeptide TomM having an amino acid sequence of SEQ ID NO:4, a thirdsequence encoding a polypeptide TomN having an amino acid sequence ofSEQ ID NO:5, a fourth sequence encoding a polypeptide TomO having anamino acid sequence of SEQ ID NO:6, and a fifth sequence encoding apolypeptide TomP having an amino acid sequence of SEQ ID NO:7, and thefirst to fifth sequences are aligned so that expressed TomL-TomP canform an active monooxygenase protein;

[0024] wherein the promoter is linked to the DNA fragment in a mannerallowing expression of the toluene monooxygenase protein encoded by theDNA fragment.

[0025] According to still another aspect of the present invention, thereis provided a recombinant DNA comprising a vector; a promoter; and a DNAfragment containing a region encoding a toluene monooxygenase, where theregion comprises a first sequence encoding a polypeptide TomL having anamino acid sequence of SEQ ID NO:3, a second sequence encoding apolypeptide TomM having an amino acid sequence of SEQ ID NO:4, a thirdsequence encoding a polypeptide TomN having an amino acid sequence ofSEQ ID NO:5, a fourth sequence encoding a polypeptide TomO having anamino acid sequence of SEQ ID NO:6, and a fifth sequence encoding apolypeptide TomP having an amino acid sequence of SEQ ID NO:7, and thefirst to fifth sequences are aligned so that expressed TomL-TomP canform an active monooxygenase protein,

[0026] wherein one or more nucleotides have been deleted from,substituted in, and/or added to at least one of the sequences of the DNAfragment with the proviso that the protein does not loose toluenemonooxygenase activity,

[0027] wherein the promoter and the DNA fragment are functionally linkedin a manner enabling expression of the toluene monooxygenase proteinencoded by the DNA fragment.

[0028] According to still another aspect of the present invention, thereis provided a recombinant DNA comprising a vector; a first promoter anda first DNA fragment functionally linked thereto; and a second promoterand a second DNA fragment functionally linked thereto; wherein the firstDNA fragment contains a region encoding a polypeptide TomK having anamino acid sequence of SEQ ID NO:2 wherein the function of TomK is toenhance a toluene monooxygenase activity of a protein consisting atleast of TomL to TomP, or encoding a variant TomK having an amino acidsequence varied from SEQ ID NO:2 with the proviso that the function ofTomK is not impaired; the second DNA fragment contains a region encodinga toluene monooxygenase, where the region comprises a first sequenceencoding a polypeptide TomL having an amino acid sequence of SEQ IDNO:3, a second sequence encoding a polypeptide TomM having an amino acidsequence of SEQ ID NO:4, a third sequence encoding a polypeptide TomNhaving an amino acid sequence of SEQ ID NO:5, a fourth sequence encodinga polypeptide TomO having an amino acid sequence of SEQ ID NO:6, and afifth sequence encoding a polypeptide TomP having an amino acid sequenceof SEQ ID NO:7, and the first to fifth sequences are aligned so thatexpressed TomL-TomP can form an active monooxygenase protein, whereinone or more nucleotides have been deleted from, substituted in, and/oradded to at least one of the sequences of the second DNA fragment withthe proviso that the protein does not loose toluene monooxygenaseactivity,

[0029] wherein the vector is linked to the DNA fragment in a mannerenabling expression of the toluene monooxygenase protein encoded by theDNA fragment.

[0030] Further, according to still another aspect of the presentinvention, there is provided a transformant obtainable by introducing arecombinant DNA comprising a vector enabling maintenance or replicationin a host and a DNA fragment of about 5.8 Kb containing a toluenemonooxygenase gene having a following restriction map, where 1 BamHIrestriction site, 2 EcoRI restriction sites, 1 HpaI restriction site, 1KpnI restriction site, 1 NcoI restriction site, 1 NspV restriction site,1 SacI restriction site, 2 SmaI restriction sites, 3 SphI restrictionsites, 2 XhoI restriction sites, no ClaI restriction site, no DraIrestriction site, no EcoRV restriction site, no HindIII restrictionsite, no NdeI restriction site, no NheI restriction site, no PvuIIrestriction site, no ScaI restriction site, no Sse8387I restrictionsite, no StuI restriction site, and no XbaI restriction site arepresent.

[0031] Further, according to still another aspect of the presentinvention there is provided a transformant obtainable by introducing arecombinant DNA into a host microorganism, where the recombinant DNAcomprises a vector enabling maintenance or replication in a host, and aDNA fragment ligated thereto having the nucleotide sequence of SEQ IDNO:1 with deletion, substitution and/or addition of one or more bases,still encoding an active toluene monooxygenase.

[0032] Further, according to still another aspect of the presentinvention, there is provided a transformant obtainable by introducing arecombinant DNA comprising a vector, a promoter and a DNA fragment intoa host microorganism where the DNA fragment contains a region encoding atoluene monooxygenase, where the region comprises a first sequenceencoding a polypeptide TomL having an amino acid sequence of SEQ IDNO:3, a second sequence encoding a polypeptide TomM having an amino acidsequence of SEQ ID NO:4, a third sequence encoding a polypeptide TomNhaving an amino acid sequence of SEQ ID NO:5, a fourth sequence encodinga polypeptide TomO having an amino acid sequence of SEQ ID NO:6, and afifth sequence encoding a polypeptide TomP having an amino acid sequenceof SEQ ID NO:7, and the first to fifth sequences are aligned so thatexpressed Tom L-TomP can form an active monooxygenase protein;

[0033] wherein the promoter and the DNA fragment are functionally linkedin a manner enabling expression of the toluene monooxygenase proteinencoded by the DNA fragment.

[0034] According to still another aspect of the present invention, thereis provided a method for producing a toluene monooxygenase, whichcomprises a step of making the transformant according to any one of theembodiment of the present invention mentioned above to produce thetoluene monooxygenase being a gene product of the recombinant DNAintroduced in the transformant.

[0035] According to still another aspect of the present invention, thereis provided a method for degrading at least either of a chlorinatedaliphatic hydrocarbon compound or an aromatic compound, which comprisesa step of degrading at least either of the chlorinated aliphatichydrocarbon compound or aromatic compound using the transformantaccording to any one of the aspects of the present invention mentionedabove.

[0036] According to still another aspect of the present invention, thereis provided a method for cleaning a medium contaminated with at leasteither of a chlorinated aliphatic hydrocarbon compound or an aromaticcompound, which comprises a step of degrading at least either of achlorinated aliphatic hydrocarbon compound or an aromatic compound usingthe transformants according to any one of the aspects of the presentinvention mentioned above.

[0037] According to still another aspect of the present invention, thereis provided a method of remedying an environment polluted with at leasteither of a chlorinated aliphatic hydrocarbon compound or an aromaticcompound as a pollutant, comprising a step of degrading the pollutantsusing the transformant according to any one of the aspects of thepresent invention mentioned above.

[0038] According to still another aspect of the present invention, thereis provided a component polypeptide having any one of amino acidsequences of SEQ ID Nos:2-8, which can constitute a toluenemonooxygenase.

[0039] According to still another aspect of the present invention, thereis provided a toluene monooxygenase comprising at least componentpolypeptides TomL-TomP of amino acid sequences of SEQ ID NOs: 3-7.

[0040] According to still another aspect of the present invention, thereis provided a variant toluene monooxygenase comprising at leastcomponent polypeptides TomL-TomP of amino acid sequences of SEQ IDNos.:3-7 wherein one or more amino acids have been deleted from,substituted to, and/or added to the polypeptides with the proviso thatthe toluene monooxygenase does not loose its activity.

BRIEF DESCRIPTION OF THE DRAWINGS

[0041]FIG. 1 shows a restriction map of a DNA fragment of about 5.8 Kbcarrying a toluene monooxygenase gene;

[0042]FIG. 2 is comprised of FIGS. 2A, 2B, 2C, 2D, 2E, 2F, 2G, 2H, 2I,2J, 2K, 2L, 2M, 2N, 2O, 2P, 2Q and 2R showing a nucleotide sequence of atoluene monooxygenase gene of FERM BP-4235;

[0043]FIG. 3 is an amino acid sequence (TomK) encoded by a region tomKin the nucleotide sequence of FIG. 2;

[0044]FIG. 4 is comprised of FIGS. 4A, 4B and 4C showing an amino acidsequence (TomL) coded by a region tomL in the nucleotide sequence ofFIG. 2;

[0045]FIG. 5 is an amino acid sequence (TomM) coded by a region tomM inthe nucleotide sequence of FIG. 2;

[0046]FIG. 6 is comprised of FIGS. 6A, 6B, 6C and 6D showing an aminoacid sequence (TomN) coded by a region tomN in the nucleotide sequenceof FIG. 2;

[0047]FIG. 7 is an amino acid sequence (TomO) coded by a region tomO inthe nucleotide sequence of FIG. 2;

[0048]FIG. 8 is comprised of FIGS. 8A, 8B and 8C showing an amino acidsequence (TomP) coded by a region tomP in the nucleotide sequence ofFIG. 2;

[0049]FIG. 9 is an amino acid sequence (TomQ) coded by a region tomQ inthe nucleotide sequence of FIG. 2;

[0050]FIG. 10 is a nucleotide sequence of a first primer employed inExample 6;

[0051]FIG. 11 is a nucleotide sequence of a second primer employed inExample 6;

[0052]FIG. 12 is a nucleotide sequence of a third primer employed inExample 6;

[0053]FIG. 13 is a nucleotide sequence of a fourth primer employed inExample 6;

[0054]FIG. 14 is a nucleotide sequence of a fifth primer employed inExample 6; and

[0055]FIG. 15 shows time-course changes in TCE in the gas phase inExample 3.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0056] The DNA fragment containing a toluene monooxygenase geneaccording to the present invention is isolated from Burkholderia cepaciastrain KK01 (FERM BP-4235, hereinafter referred to as Strain KK01). Themicrobiological characteristics and culture conditions of Strain KK01are as follows (see Japanese Patent Application Laid-Open No. 6-22769).Strain KK01

[0057] Morphological characteristics

[0058] (1) Gram staining: Negative

[0059] (2) Size and shape: Rod of 1.0-2.0 μm in length and 0.5 μm inwidth

[0060] (3) Motility: Motile

[0061] B. Growth on various culture media Medium Growth temperature (°C.) Growth Blood agar medium 37 + Lactose agar medium 37 + Chocolateagar medium 37 ++ GMA 37 − Scylo 37 − Standard agar medium 4 − Standardagar medium 25 ± Standard agar medium 37 + Standard agar medium 41 ±

[0062] C. Physiological characteristics

[0063] (1) Aerobic or anaerobic: Obligate aerobic

[0064] (2) Sugar degradation mode: Oxidation

[0065] (3) Oxidase production: +

[0066] (4) Silver nitrate reduction: +

[0067] (5) Hydrogen sulfide production: −

[0068] (6) Indole production: −

[0069] (7) Urease production: −

[0070] (8) Gelatin liquefaction: −

[0071] (9) Arginine hydrolysis: −

[0072] (10) Lysine decarboxylation: +

[0073] (11) Ornithine decarboxylation: −

[0074] (12) Utilization of citric acid: +

[0075] (13) Methyl carbinol acetyl reaction (VP reaction):

[0076] (14) Detection of tryptophan deaminase: −

[0077] (15) ONPG:

[0078] (16) Assimilation of carbohydrates

[0079] Glucose: +

[0080] Fructose: +

[0081] Maltose: +

[0082] Galactose: +

[0083] Xylose: +

[0084] Mannitol: ±

[0085] Sucrose: −

[0086] Lactose: +

[0087] Esculin: −

[0088] Inositol: −

[0089] Sorbitol: −

[0090] Rhamnose: −

[0091] Melibiose: −

[0092] Amygdalin: −

[0093] L-(+)-arabinose: +

[0094] Isolation of the DNA fragment according to the present inventionis achieved by partial digestion of the total DNA of strain KK01 with arestriction enzyme Sau3AI. Specifically, total DNA can be prepared bythe standard method, in which the above microorganism is grown in asuitable medium, for example, LB medium (containing 10 g of trypton, 5 gof yeast extract, and 5 g of sodium chloride in 1 litter) and then cellsare disrupted, for example, in the presence of sodium dodecyl sulfate(SDS) at 70° C. The total DNA is then partially digested by Sau3AI toobtain a DNA fragment of about 5.8 Kb carrying a toluene monooxygenasegene. The DNA fragment thus obtained is ligated to a plasmid vectorcompletely digested by BamHI, for example, pUC18, and the recombinantvector is introduced into competent cells of, for example, E. coliJM109, prepared by the Hanahan method to obtain transformants. Then,transformants can be selected by a suitable method, for example, byculturing cells on an LB medium plate containing ampicillin.

[0095] In order to select a transformant containing a recombinant vectorcarrying a toluene monooxygenase gene from the above transformants, itis preferable to add cresol, phenol, or the like to LB medium fortransformant selection in advance. The transformant carrying a toluenemonooxygenase gene can be selected as brown colonies, since thesesubstrates are monooxygenated by toluene monooxygenase to producemethylcatechol or catechol which is then autooxidized to develop color.Alternatively, after culturing cells on an ordinary LB medium plate,various substrates may be sprayed onto the plate to select browncolonies in a similar manner.

[0096] The isolated DNA fragment of about 5.8 Kb has the followingrestriction sites: Restriction Number of enzyme restriction sites BamHI1 EcoRI 2 HpaI 1 KpnI 1 NcoI 1 NspV 1 SacI 1 SimI 2 SphI 3 XhoI 2

[0097] The DNA fragment has no ClaI, DraI, EcoRV, HindIII, NdeI, NheI,PvuII, ScaI, Sse83871, StuI, or XbaI restriction site.

[0098] The restriction map of the DNA fragment of the present inventionis as shown above. Toluene monooxygenase genes derived from Burkholderiacepacia G4 5223 PR1 (U.S. Pat. No. 5543317), derived from Burkholderiasp. JS150 (Appl. Environ. Microbiol., 61, 3336 (1995), derived fromPseudomonas pickettii PK01 (J. Bacteriol., 176, 3749 (1994)), andderived from Pseudomonas mendocina KR1 (J. Bacteriol., 173, 3010 (1991))were reported. Phenol hydroxylases reported to have a similar structureare derived from Acinetobacter calcoaceticus NCIIB8250 (Mol. Microbiol.,18, 13 (1995)), Pseudomonas sp. CF600 (J. Bacteriol., 172, 6826 (1990)),Pseudomonas spp. (J. Bacteriol., 177, 1485 (1995)), and Pseudomonasputida P35X (Gene, 151, 29 (1994)). The DNA fragment of the presentinvention has, however, a restriction map different from any of those.It is thus clear that the DNA fragment of the present invention containsa novel toluene monooxygenase gene.

[0099] Although the DNA fragment thus obtained can sufficiently enablesthe degradation of aromatic compounds and/or chlorinated aliphatichydrocarbon compounds even in pUC18, it can be integrated in anexpression vector or a vector of a wide host range to improve thedegradation ability or to be optimized for the treatment site.

[0100] The plasmid according to the present invention can be constructedfrom following elements:

[0101] 1) Toluene monooxygenase gene;

[0102] 2) Marker gene (drug-resistance, auxotrophic complement, or thelike); and

[0103] 3) Vector containing an autonomous replication sequence (plasmid,or the like).

[0104] As the toluene monooxygenase gene, the DNA fragment of about 5.8kb as shown above can be employed by itself, or a constitutioncontaining elements necessary for a toluene monooxygenase activity canbe also employed, for example, with or without spacer sequences.Further, each element can be varied with the proviso that its functionis not impaired. These variations can be attained by changing DNAsequences encoding them.

[0105] As the drug-resistance genes, an ampicillin resistance gene, akanamycin (G418, neomycin) resistance gene, a tetracycline resistancegene, a chloramphenicol resistance gene, a hygromycin resistance genecan be employed. For auxotrophic complement, a gene sequence to supplythe nutrient required by the host organism is used. Typically, a geneenabling the synthesis of the required amino acid is utilized.

[0106] As the autonomous replication sequences, a sequence derived fromplasmid RSF1010, which can function as a wide host range replicationregion in most of the gram-negative bacteria, can be employed. It can bealso employed vector pBBR122 (Mo Bi Tec) containing a wide host rangereplication region which does not belong to any incompatible groups,IncP, IncQ, or IncW or the like.

[0107] For the recombinant plasmid according to the present invention,various promoters and terminators can be employed and various factorscan be further introduced to improve and control the ability ofdegrading aromatic compounds and/or chlorinated aliphatic hydrocarboncompounds. Specifically, promoters such as lac, trc, tac, T3, and T7canbe employed. As a terminator, a rrnB operon terminator or the like canbe employed. Also, introduction of a repressor gene such as lacIq and alac operator enables expression control with an inducer such asisopropyl thiogalactoside (IPTG). Alternatively, the absence of thesesuppressor and operator as elements, enables constitutive expression ofdegradation activity. In addition, a temperature-sensitive controlsystem or the like can be employed.

[0108] For recombination of a DNA fragment containing the toluenemonooxygenase gene into an expression vector containing these regulatingelements, natural restriction sites can be utilized as it is, orrestriction sites may be newly created by site-directed mutagenesis or apolymerase chain reaction using a primer involving base substitution. Ingeneral, recombination into an expression vector often utilizes NcoIrestriction sites. It is convenient to design so as to create an NcoIrestriction site in the initiation codon ATG or GTG region bysite-directed mutagenesis or primer design. Known methods using anadaptor can be employed. For optimization of expression, the DNAfragment may be properly deleted using exonuclease III or Bal3lnuclease. As described above, molecular biological techniques suitablefor the purpose can be employed for recombination into an expressionvector.

[0109] As a method for introducing the recombinant plasmid carrying adesired gene into a host organism, any methods that can introduce aforeign gene into a host can be employed, and known methods, forexample, the calcium chloride method, the electroporation method, andthe conjugation transfer method can be employed.

[0110] In the present invention, any microorganisms can be used as ahost organism so long as it can express the aromatic compounds and/orchlorinated aliphatic hydrocarbon compounds-degrading activity after theintroduction of the recombinant plasmid, including the generaEscherichia, Pseudomonas, Burkholderia, Acinetobacter, Moraxella,Alcaligenes, Vibrio, Nocardia, Bacillus, Lactobacillus, Achromobacter,Arthrobacter, Micrococcus, Mycobacterium, Methylosinus, Methylomonas,Welchia, Methylocystis, Nitrosomonas, Saccharomyces, Candida,Torulopsis, and Ralstonia.

[0111] In addition, the aromatic compounds and/or chlorinated aliphatichydrocarbon compounds-degrading microorganisms such as strain J1, strainJM1, Pseudomonas sp. strain TL1, strain KK01, Pseudomonas alcaligenesstrain KB2, Alcaligenes sp. strain TL2, and Vibrio sp. strain KB1 can beemployed as a host. These strains have been deposited in the NationalInstitute of Bioscience and Human Technology, Agency of IndustrialScience and Technology of Japan. The date of deposit, Accession No., andmicrobiological characteristics of these strains other than the strainKK01 already described are shown below.

[0112] Strain J1 (Deposit date: May 25, 1994, Accession No. FERMBP-5102)

[0113] A. Morphological characteristics

[0114] Gram staining: Positive

[0115] Size and shape of cells: Polymorphous rod of 1-6

[0116] μm in length and about 0.5-2 μm in width

[0117] Mobility: Negative

[0118] Colony: Cream to light pink, sticky

[0119] B. Growth on various media

[0120] BHIA: Good growth

[0121] MacConkey: No growth

[0122] C. Optimal temperature for growth: 25° C.>30° C.>35° C.

[0123] D. Physiological characteristics

[0124] Aerobic or anaerobic: aerobic

[0125] TSI (slant/butt): Alkaline/alkaline, H₂S (−)

[0126] Oxidase: Negative

[0127] Catalase: Positive

[0128] Sugar fermentation

[0129] Glucose: Negative

[0130] Sucrose: Negative

[0131] Raffinose: Negative

[0132] Galactose: Negative

[0133] Maltose: Negative

[0134] Urease: Positive

[0135] Esculin: Positive

[0136] Nitric acid: Negative

[0137] Strain JM1 (Deposit date: Jan. 10, 1995, Accession No. FERMBP-5352)

[0138] Gram staining and morphology: Gram-negative rod

[0139] Growth on various media

[0140] BHIA: Good growth

[0141] MacConkey: Possible to grow

[0142] Colony color: Cream

[0143] Optimal temperature for growth: 25° C.>30° C.>35° C.

[0144] Mobility: Negative (semi-fluid medium)

[0145] TSI (slant/butt): Alkaline/alkaline, H₂S (−)

[0146] Oxidase: Positive (weak)

[0147] Catalase: Positive

[0148] Sugar fermentation

[0149] Glucose: Negative

[0150] Sucrose: Negative

[0151] Raffinose: Negative

[0152] Galactose: Negative

[0153] Maltose: Negative

[0154] Urease: Positive

[0155] Esculin hydrolysis (β-glucosidase): Positive

[0156] Nitrate reduction: Negative

[0157] Indole production: Negative

[0158] Glucose acidification: Negative

[0159] Arginine dehydrase: Negative

[0160] Gelatin hydrolysis (protease): Negative

[0161] β-Galactosidase: Negative

[0162] Assimilation of compounds

[0163] Glucose: Negative

[0164] L-Arabinose: Negative

[0165] D-Mannose: Negative

[0166] D-Mannitol: Negative

[0167] N-Acetyl-D-glucosamine: Negative

[0168] Maltose: Negative

[0169] Potassium gluconate: Negative

[0170] n-Capric acid: Positive

[0171] Adipic acid: Negative

[0172] dl-Malic acid: Positive

[0173] Sodium citrate: Positive

[0174] Phenyl acetate: Negative

[0175] Strain J1 is an aromatic compound-assimilating bacterium whichdegrades organic chlorinated compounds with the participation ofoxygenase. In spite of its excellent ability of degrading organicchlorinated compounds that it can almost completely degrade about 20 ppmof TCE at a low temperature of 15° C. close to natural environment suchas soil, it requires aromatic compounds such as phenol, toluene, andcresol as a degradation inducer. Strain JM1 has the same microbiologicalcharacteristics as the parental strain J1 except that it can degradeorganic chlorinated compounds in the absence of aromatic compounds suchas phenol, toluene, and cresol as a degradation inducer.

[0176] Strain TL1 (Deposit date: Jan. 10, 1995, Deposit No. FERMP-14726/FERM BP-6923.

[0177] A. Gram staining and morphology: Gram-negative rod

[0178] B. Growth on various media

[0179] Standard agar: Good growth

[0180] MacConkey agar: Poor growth

[0181] C. Optimal temperature for growth: 25° C.>35° C.

[0182] D. Physiological characteristics

[0183] Aerobic/anaerobic: Aerobic

[0184] TSI (slant/butt): Alkaline/alkaline, H₂S (−)

[0185] Oxidase: Positive

[0186] Catalase: Positive

[0187] Oxidation/fermentation test: −/−

[0188] Potassium nitrate reduction: Negative

[0189] Indole production from L-tryptophan: Negative

[0190] Glucose acidification: Negative

[0191] Arginine dehydrase: Negative

[0192] Urease: Negative

[0193] Esculin hydrolysis (β-glucosidase): Negative

[0194] Gelatin hydrolysis (protease): Negative

[0195] β-Galactosidase: Negative

[0196] Cytochrome oxidase: Positive

[0197] E. Assimilation of sugars, organic acids, etc.

[0198] Glucose: Positive

[0199] L-Arabinose: Positive

[0200] D-Mannose: Negative

[0201] D-Mannitol: Positive

[0202] N-Acetyl-D-glucosamine: Negative

[0203] Maltose: Negative

[0204] Potassium gluconate: Positive

[0205] n-Capric acid: Negative

[0206] Adipic acid: Positive

[0207] dl-Malic acid: Negative

[0208] Sodium citrate: Negative

[0209] Phenyl acetate: Negative

[0210] Strain TL2 (Deposit date on Nov. 15, 1994, Deposit No. FERMP-14642/FERM BP-6913.

[0211] A. Gram staining and morphology: Gram-negative rod

[0212] B. Growth on various media

[0213] Standard agar: Good growth

[0214] MacConkey agar: Poor growth

[0215] C. Optimal temperature for growth: 25° C.>35° C.

[0216] D. Physiological characteristics

[0217] Aerobic/anaerobic: Aerobic

[0218] TSI (slant/butt): Alkaline/alkaline, H₂S (−)

[0219] Oxidase: Positive

[0220] Catalase: Positive

[0221] Oxidation/fermentation test: −/−

[0222] Potassium nitrate reduction: Positive

[0223] Indole production from L-tryptophan: Negative

[0224] Glucose acidification: Negative

[0225] Arginine dehydrase: Negative

[0226] Urease: Negative

[0227] Esculin hydrolysis (β-glucosidase): Negative

[0228] Gelatin hydrolysis (protease): Negative

[0229] β-Galactosidase: Negative

[0230] Cytochrome oxidase: Positive

[0231] E. Assimilation of sugars, organic acids, etc.

[0232] Glucose: Negative

[0233] L-Arabinose: Negative

[0234] D-Mannose: Negative

[0235] D-Mannitol: Negative

[0236] N-Acetyl-D-glucosamine: Negative

[0237] Maltose: Negative

[0238] Potassium gluconate: Positive

[0239] n-Capric acid: Positive

[0240] Adipic acid: Positive

[0241] dl-Malic acid: Positive

[0242] Sodium citrate: Positive

[0243] Phenyl acetate: Positive

[0244] Strain KB1 (Deposit date: Nov. 15, 1994, Deposit No. FERMP-14643/FERM BP-6914.

[0245] A. Gram staining and morphology: Gram-negative bacillus

[0246] B. Growth conditions on various media

[0247] Standard agar: Good growth

[0248] MacConkey agar: Good growth

[0249] C. Optimal temperature for growth: 25° C.>35° C.

[0250] D. Physiological characteristics

[0251] Aerobic/anaerobic: Aerobic

[0252] TSI (slant/butt): Alkaline/alkaline, H2 S(−)

[0253] Catalase: Positive

[0254] Oxidation/fermentation test: −/−

[0255] Potassium nitrate reduction: Positive

[0256] Indole productivity from L-tryptophan: Negative

[0257] Glucose acidification: Negative

[0258] Arginine dehydrase: Positive

[0259] Urease: Positive

[0260] Esculin hydrolysis (β-glucosidase): Negative

[0261] Gelatin hydrolysis (protease): Negative

[0262] β-Galactosidase: Negative

[0263] Cytochrome oxidase: Positive

[0264] E. Assimilation of sugars, organic acids, etc.

[0265] Glucose: Negative

[0266] L-Arabinose: Negative

[0267] D-Mannose: Negative

[0268] D-Mannitol: Negative

[0269] N-Acetyl-D-glucosamine: Positive

[0270] Maltose: Negative

[0271] Potassium gluconate: Positive

[0272] n-Capric acid: Positive

[0273] Adipic acid: Positive

[0274] dl-Malic acid: Positive

[0275] Sodium citrate: Negative

[0276] Phenyl acetate: Positive

[0277] Strain KB2 (Deposit date: Nov. 15, 1994, Accession No. FERMBP-5354)

[0278] A. Gram staining and morphology: Gram-negative rod

[0279] B. Growth on various media

[0280] Standard agar: Good growth

[0281] MacConkey agar: Good growth

[0282] C. Optimal temperature for growth: 25° C.>35° C.

[0283] Growth at 42° C.: Good

[0284] D. Physiological characteristics

[0285] Aerobic/anaerobic: Aerobic

[0286] TSI (slant/butt): Alkaline/alkaline, H₂S (−)

[0287] Catalase: Positive

[0288] Oxidation/fermentation test: −/−

[0289] Potassium nitrate reduction: Positive

[0290] Indole production from L-tryptophan: Negative

[0291] Glucose acidification: Negative

[0292] Arginine dehydrase: Negative

[0293] Urease: Negative

[0294] Esculin hydrolysis (β-glucosidase): Negative

[0295] Gelatin hydrolysis (protease): Negative

[0296] β-Galactosidase: Negative

[0297] Cytochrome oxidase: Positive

[0298] E. Assimilation of sugars, organic acids, etc.

[0299] Glucose: Negative

[0300] L-Arabinose: Negative

[0301] D-Mannose: Negative

[0302] D-Mannitol: Negative

[0303] N-Acetyl-D-glucosamine: Negative

[0304] Maltose: Negative

[0305] Potassium gluconate: Positive

[0306] n-Capric acid: Negative

[0307] Adipic acid: Positive

[0308] dl-Malic acid: Positive

[0309] Sodium citrate: Negative

[0310] Phenyl acetate: Negative

[0311] Further, in order to exploit the microbial degrading ability moreeffectively, it is preferable to select the host microorganism forrecombinants from the microorganisms isolated to the environment to betreated, more preferably a dominant microorganism in the environment,considering environmental adaptation of the recombinant. Generally, inthe natural world, microorganisms that have existed in an environmentwill adapt to the environment most probably, and the probability of thesurvival of foreign microorganisms introduced into the environment isnot high. On the other hand, when a very strong microorganism isintroduced from outside, it may disturb the existing ecosystem. Thus,the use of the indigenous microorganisms as a host is a superior methodin environmental adaptability, survival, and safety.

[0312] A transformant to which a recombinant plasmid has been introducedmay be cultured in the conditions suitable for the growth of the host.For example, a carbon and nitrogen source such as yeast extract,trypton, and peptone, and a inorganic salt such as sodium chloride andpotassium chloride can be used. An M9 medium (containing 6.2 g ofNa₂HPO₄, 3.0 g of KH₂PO₄, 0.5 g of NaCl, and 1.0 g of NH₄Cl in 1 litter)supplemented with various minerals and suitable carbon sources such assodium malate, sodium succinate, sodium lactate, sodium pyruvate, sodiumglutamate, sodium citrate, etc. can also be employed. Further, yeastextract, trypton, peptone, etc. can be used in combination. The pH ofthe growth medium and culture temperature can be adjusted to thosesuitable for the host microorganism, although pH of about 5-9 andculture temperature of 15-37° C. are generally preferable.

[0313] A transformant containing a recombinant DNA carrying a toluenemonooxygenase gene can be suitably employed for the treatment to degradechlorinated aliphatic hydrocarbon compounds and aromatic compounds(hereinafter referred to as “pollution compounds”) contained in amedium. In other words, the degradation treatment for the pollutioncompounds according to the present invention can be carried out bybringing the transformant into contact with the pollution compounds inan aqueous medium, soil, or a gas phase. Any method can be used tocontact the degrading microorganisms with the pollution compounds solong as the microorganisms can express the degrading activity. Variousmethods such as a batch method, semi-continuous method, and continuousmethod can be employed. Microorganisms semi-immobilized or immobilizedon an appropriate carrier can be also used. The subject such as pollutedwater, drainage, waste water, soil, and gas phase can be treated byvarious methods, as required. These treatment methods are describedbelow.

[0314] The degradation treatment of the pollution compounds in anaqueous medium according to the present invention can be carried out bycontacting the degrading microorganism with the pollution compounds inthe aqueous medium. The representative treating methods are describedbelow. However, the method according to the present invention is notlimited thereto, but applicable for any clean-up of the pollutioncompounds in an aqueous medium.

[0315] The simplest method is, for example, to introduce the degradingmicroorganism directly into an aqueous medium contaminated with thepollution compounds. In this case, it is preferable to optimize the pH,salt concentrations, temperature, and pollutant concentrations of theaqueous medium according to the degrading microorganism.

[0316] As another application mode, the degrading microorganism is grownin a culture vessel, and an aqueous medium containing the pollutioncompounds is introduced into the vessel at a predetermined flow rate todegrade these compounds. The aqueous medium can be introduced anddischarged continuously, intermittently or batch-wise according to thetreatment capacity. It is preferable to optimize the system by a systemcontrol in accordance to the concentrations of the pollution compounds.

[0317] Alternatively, the degrading microorganism may be first attachedto a carrier such as soil particles and the filled in a reactor vessel,to which an aqueous medium containing the pollution compounds isintroduced for degradation treatment. In this case, any carrier can beemployed not restricted to soil particles, but carriers having a highcapacity to retain microorganisms and not preventing aeration arepreferable. To provide the microorganism with habitats, it can be usedvarious bioreactor carriers, for example, those conventionally employedin the pharmaceutical industry, food industry, and wastewater treatmentsystems. More specifically, there can be used inorganic particulatecarries such as porous glass, ceramics, metal oxides, activated carbon,kaolinite, bentonite, zeolite, silica gel, alumina, and anthracite; gelcarries such as starch, agar, chitin, chitosan, polyvinyl alcohol,alginic acid, polyacrylamide, carrageenan, and agarose; ion-exchangecellulose, ion-exchange resins, cellulose derivatives, glutaraldehyde,polyacrylic acid, polyurethane, polyester, or the like. As naturalmaterials, cellulose materials such as cotton, hemp, and papers, andlignin materials such as saw dust and barks can be employed.

[0318] The degradation treatment of the pollution compounds in soilaccording to the present invention can be carried out by bringing thedegrading microorganism in contact with the pollution compounds in thesoil. The representative treating methods are described below. However,the method according to the present invention is not limited thereto butapplicable to any clean-up of the pollution compounds in soil.

[0319] The simplest method is, for example, to introducing degradingmicroorganisms directly into the soil polluted with the pollutioncompounds. Introduction of the microorganism may be carried out byspraying it on the surface of the soil and, when the treatment extendsto deep underground, by introducing it through the well arranged in theunderground, wherein the application of pressure of air, water, etc.allows the microorganism to spread over the wide area of the soil andmakes the process more effective. In this case, it is necessary toadjust various conditions of the soil so that they are suitable for themicroorganism used for the process.

[0320] Another use is such that first the microorganism is attached to acarrier, next the carriers are charged into the reaction vessel, andthen the reaction vessel is introduced into, primarily, the aquifer ofthe contaminated soil, to undergo degradation treatment.

[0321] The form of the reaction vessel is desirably like a fence or afilm which can cover the wide area of the soil. Any carrier can be used,but it is preferable to use those having an excellent retention ofmicroorganisms and not inhibiting aeration. As a material of thecarrier, which can provide suitable habitats for microorganisms, forexample, it can be used various bioreactor carriers, for example, thoseconventionally employed in the pharmaceutical industry, food industry,and wastewater treatment systems.

[0322] According to the present invention, the degradation treatment ofthe pollution compounds in gas phase can be achieved by contacting themicroorganism with the contaminants in the gas phase. The representativemodes are shown below, but are not intended to limit the presentinvention. The present invention is applicable to purification treatmentof any gas phase contaminated with the pollution compounds.

[0323] One mode is, for example, such that the degradation microorganismis cultured in a culture vessel, and then the gas containing thepollution compounds is introduced into the vessel at a given flow rateto undergo degradation treatment. The method of introducing the gas isnot limited specifically, but it is desirably such that introduction ofthe gas causes agitation of the culture medium and promote its aeration.Introduction and discharge of the gas may be carried out continuously,or it may be carried out intermittently according to the degradationcapacity. A batch method is also applicable. Preferably such control issystematized in accordance with the concentrations of the pollutioncompounds to give optimum results.

[0324] Another mode is such that the microorganism is attached to acarrier like soil particles, next the carriers are put into a reactionvessel, and then the gas containing the pollution compounds isintroduced into the vessel to undergo degradation treatment. Besidesparticles of soil, any carrier can be used, however, it is desirable touse those having an excellent retention of microorganisms and notinhibiting aeration. As a material of the carrier, which can providesuitable habitats for microorganisms, for example, it can be usedvarious bioreactor carriers, for example, those conventionally employedin the pharmaceutical industry, food industry, and wastewater treatmentsystems.

[0325] As materials which can retain the degrading microorganism andsupply it with nutrient, many examples can be found in the compost usedin the agriculture, forestry and fisheries. Specifically, dry materialsfrom plants, such as straw of grains, sawdust, rice bran, bean curdlees, bagasse and so on, and seafood wastes, such as shells of crabs andlobster and so on are applicable.

[0326] In purification of contaminated gas, the degrading microorganismmay be introduced after the carrier material is packed. To make thedegradation reaction efficient, it is preferable that theabove-mentioned nutrient, water content, oxygen concentration, etc. arekept in desirable conditions. The ratio of the carrier to water in areaction vessel may be determined considering the growth of themicroorganism and aeration. The shape of the vessel may be selectedconsidering the amount and concentration of the gas undergoingtreatment, but preferably it is designed to enhance the contact of thegas with the microorganism held on the carrier. For example, column,tube, tank and box type are applicable. The vessel of these forms may bejoined together with an exhaust duct and a filter to form one unit, orplural vessels may be connected according to the capacity.

[0327] Contaminated gas is sometimes adsorbed by the carrier material inthe beginning of the reaction and there is very few case where theeffect of utilizing microorganism may not be exhibit. After a certainperiod of time, however, it is thought that the contaminants adhered tothe carrier material is degraded, and further contaminants can beadsorbed by the surface of the material to restore adsorption of thematerial. Thus, a constant decomposition rate is expected withoutsaturation of the pollutant-eliminating ability.

[0328] The method according to the present invention is applicable forthe treatment of waste liquid, soil and air in a closed system or opensystem. Moreover, microorganisms may be immobilized on a carrier, orvarious methods promoting their proliferation may be employed incombination.

[0329] The present invention is explained more specifically by means ofthe following examples.

EXAMPLE 1 Cloning of toluene monooxygenase gene of strain KK01

[0330] Cells of strain KK01 (FERM BP-4235) which can assimilate toluenewere cultured in 100 ml of LB medium (containing 10 g of trypton, 5 g ofyeast extract, and 5 g of sodium chloride in 1 liter) overnight,harvested and washed with 100 mM phosphate buffer (pH 8.0). To the cellsthus obtained, 10 ml of STE (10 mM tris (pH 8.0)/1 mM EDTA/100 mM sodiumchloride) and 1 ml of 10% sodium dodecyl sulfate (final concentration ofabout 1%) were added. After the cells were incubated at 70° C. for 30minutes for lysis, phenol treatment and ethanol sedimentation werecarried out. DNA thus obtained was dissolved in a 10 mM tris (pH 8.0)/1mM EDTA buffer (TE).

[0331] The DNA thus obtained was dissolved at various concentrations andtreated with a restriction enzyme Sau3AI (Takara Shuzo Co., Ltd.) at 37°C. for 15 minutes for partial digestion. Aliquots of the partialdigestion products were applied to gel electrophoresis on 0.8% agarosegel to identify the samples almost digested to about 5-10 kb. Thesesamples were applied to spin column HR-400 (Amarsham-Pharmacia) topurify DNA fragments.

[0332] The DNA fragments were ligated to plasmid pUC18 (Takara ShuzoCo., Ltd.) completely digested with a restriction enzyme BamHI (TakaraShuzo Co., Ltd.) and dephosphorylated with BAP (Takara Shuzo Co., Ltd.),using DNA Ligation Kit Ver. 2 (Takara Shuzo Co., Ltd.). Recombinantplasmids thus prepared were then introduced into the host E. coli HB101(Takara Shuzo Co., Ltd.), and the cells were cultured on LB agar platescontaining 100 μg/ml of ampicillin as a selection agent and 200 ppmphenol as an indicator for toluene monooxygenase activity. About 15,000colonies of transformants grew on the plates.

[0333] Eight brown colonies were found in these colonies and picked up.Recombinant plasmid DNA carrying toluene monooxygenase gene wasextracted from the cells of each brown colony and the restriction mapthereof was determined. It was found that all recombinant plasmidsderived from the 8 colonies had a common insertion fragment of 5.8 kb. Aplasmid containing only the common fragment of 5.8 kb was designated aspKK01 and a restriction map of the inserted DNA fragment was made (SeeFIG. 1). A recombinant E. coli HB101 carrying a plasmid containing a 8.5kb insertion fragment containing this common 5.8 kb fragment wasdeposited in the National Institute of Bioscience and Human Technology,Agency of Industrial Science and Technology in accordance with theBudapest Treaty under the accession No. FERM BP-6916. Itsmicrobiological characteristics were identical to those of E. coli HB101except that it can degrade aromatic compounds and chlorinated aliphatichydrocarbon compounds.

[0334] In order to confirm that the inserted DNA fragment of pKK01 wasderived from strain KK01, southern hybridization was performed. DNA wasextracted from strain KK01 and completely digested with EcoRI (TakaraShuzo Co., Ltd.) or XhoI (Takara Shuzo Co., Ltd.), and then subjected tosouthern hybridization. The inserted DNA fragment of pKK01 was digestedwith BamHI-KpnI (Takara Shuzo Co., Ltd.) to obtain a DNA fragment ofabout 1.6 kb, and this was used as a probe. As a result, a strong signalwas observed around 4.3 kb with the EcoRI-digested DNA, and around 4.2kb with the XhoI digested DNA, in a good agreement with the lengths ofthe fragments predicted from the restriction map. Consequently, it wasconfirmed that the toluene monooxygenase gene contained in pKK01 wasderived from the strain KK01.

EXAMPLE 2 Monooxygenation by E. coli HB101(pKK01)

[0335] The cells of E. coli HB101(pKK01) were inoculated in 100 ml of LBmedium, cultured at 37° C. overnight, harvested, washed, and thenresuspended in 100 ml of M9 medium (6.2 g of Na₂HPO₄, 3.0 g of KH₂PO₄,0.5 g of NaCl, and 1.0 g of NH₄Cl per liter) supplemented with a mineralstock solution of the following composition (3 ml/liter of M9medium)(referred to as M9+ mineral solution). Composition of mineralstock solution Nitrilotriacetic acid 1.5 g MgSO₄ 3.0 g CaCl₂ 0.1 gNa₂MoO₄ 0.1 g FeSO₄ 0.1 g MnSO₄ 0.5 g NaCl₃ 1.0 g ZnSO₄ 0.1 g CuSO₄ 0.1g AlK(SO₄)₂ 0.1 g H₃BO₃ 0.1 g NiC1₂ 0.1 g Distilled water (to 1,000 ml)

[0336] Then, 27.5 ml vials were prepared, and 10 ml aliquot of the abovesuspension was placed in each vial, which was then tightly sealed with ateflon-coated butyl rubber stopper and aluminum seal. Gaseous toluene orbenzene was introduced into each vial with a syringe to a concentrationof 100 ppm (a concentration supposing all toluene or benzene completelydissolved in the aqueous phase in the vial). After incubation at 30° C.for 3 hours, 1 ml aliquot was taken from each vial, and cells wereremoved by centrifugation and substances of 10,000 or higher inmolecular weight were removed by ultrafiltration. Production ofortho-cresol and 3-methylcatechol from toluene and phenol and catecholfrom benzene was confirmed by HPLC, to show that toluene and benzene aremonooxygenated by toluene monooxygenase encoded by the cloned DNAfragment.

EXAMPLE 3 Degradation of aromatic compounds and chlorinated aliphatichydrocarbon compounds by E.coli HB101(pKK01)

[0337] The cells of E.coli HB101(pKK01) cultured as described in Example2 were suspended in M9 + mineral solution. Ten ml aliquots of thesuspension were placed in 27.5 ml vials. Each vial was tightly sealedwith a teflon-lined butyl rubber stopper and an aluminum seal. Gaseoustrichloroethylene (TCE), cis-1,2-dichloroethylene (cis-1,2-DCE),trans-1,2-dichloroethylene (trans-1,2-DCE), 1,1-dichloroethylene(1,1-DCE), toluene, and benzene were injected into respective vials to aconcentration of 5 ppm (a concentration supposing the introducedsubstance completely dissolved in the aqueous phase in the vial). Thevials were shaken and incubated at 30° C. The concentrations of therespective compounds in the gas phase were measured by gaschromatography after 6 hours. The results are shown in Table 1. E.coliHB101 harboring pUC18 (E.coli HB101(pUC18)) was employed as a controland degradation was evaluated in the same manner.

[0338] Another experiment was carried out on TCE degradation in the samemanner except that the initial TCE concentration was 10 ppm and when theTCE concentration in the gas phase reached about 0, the process wasrepeated for total three times. The results are shown in Table 2.

[0339] Similarly, phenol, ortho-cresol, meta-cresol and para-cresol wereintroduced into respective 27.5 ml vials each containing 10 ml of thecell suspension at a concentration of 50 ppm. Each vial was tightlysealed with a butyl rubber stopper and aluminum seal. The vials wereshaken and incubated at 30° C. The quantities of the respectivecompounds in the liquid phase were determined by the amino antipyrinemethod with a spectrophotometer to obtain their concentrations after 6hours. The results are shown in Table 2. E.coli HB101(pUC18) wasemployed as a control and degradation was evaluated in the same manner.TABLE 1 E.coliHB101 (pKK01) HB101 (pUC18) TCE 0 5.2 cis-1,2-DCE 0 4.9trans-1,2-DCE 0 5.1 1,1-DCE 0 5.3 Toluene 0 5.5 Benzene 0 4.9

[0340] TABLE 2 E.coli E.coli HB101 (pKK01) HB101 (pUC18) Phenol 0 55Ortho-cresol 0 49 Meta-cresol 0 47 Para-cresol 0 52

[0341] The above results show that E.coli HB101(pKK01) had an excellentability to degrade aromatic compounds and chlorinated aliphatichydrocarbon compounds.

EXAMPLE 4 Definition of toluene monooxygenase region

[0342] The toluene monooxygenase region was defined further bysubcloning or stepwise deletion of plasmid pKK01 obtained in Example 1,using restriction sites thereof. Toluene monooxygenase activity wasevaluated by the method in Example 3, and 5 ppm toluene was employed asa substrate.

[0343] First, a subclone pKK01 ABamHI in which a 0.7-kb fragment wasdeleted was prepared from pKK01 using the unique BamHI site at 0.7 kb.More specifically, pKK01 was completely digested by restriction enzymesBamHI and HindIII (Takara Shuzo Co., Ltd.) to obtain 2 fragments of 3.4kb and 5.1 kb. The fragments were separated by agarose gelelectrophoresis, and the 5.1 kb fragment was cut out and recovered fromthe gel and purified with a spin column HR-400 (Amarsham-Pharmacia). Thefragment was ligated to pUC18 previously completely digested by BamHIand HindIII enzymes, and E.coli HB101 was transformed with therecombinant plasmids according to the conventional method. E.coli HB101cells were then applied on an LB plate containing 100 μg/ml ofampicillin to select transformants. From the cells grown overnight in LBmedium, plasmid DNA was extracted by an alkaline method to confirm thepresence of pKK01ΔBamHI, and a transformant carrying pKK01ΔBamHI wasisolated. E.coli HB101 (pKK01ΔBamHI) cells were evaluated for toluenemonooxygenase activity. No degradation of toluene was observed,indicating that the 0.7-kb fragment is essential for toluenemonooxygenase activity.

[0344] Then, a subclone pKK01ΔEcoRI was prepared by deleting a 0.3 kbfragment from pKK01 using the 0.3 kb EcoRI restriction site of pKK01.More specifically, pKK01 was partially digested by restriction enzymeEcoRI, and then self-ligated to transform E.coli HB101. The E.coli HB101transformants were then selected on an LB plate containing 100 μg/ml ofampicillin. After the transformants were cultured in LB mediumovernight, the plasmid DNA was extracted from the cells by the alkalinemethod to confirm the presence of pKK01ΔEcoRI and a transformantcarrying pKK01ΔEcoRI was isolated. E. coli HB101(pKK01ΔEcoRI) wasevaluated for toluene monooxygenase activity. Degradation of toluene wasobserved, but the activity was lower than that of E.coli HB101(pKK101),indicating that the 0.3 kb fragment was not essential for toluenemonooxygenase activity but necessary for full expression of theactivity.

[0345] Further, the stepwise deletion method was employed to restrictthe toluene monooxygenase region from the opposite direction. Morespecifically, stepwise deletion was introduced from the XbaI restrictionsite using XbaI (Takara Shuzo Co., Ltd.) restriction site and Sse8387I(Takara Shuzo Co., Ltd.) restriction site of pUC18. The step-wisedeletion was carried out using Deletion Kit for Kilo-Sequence (TakaraShuzo Co., Ltd.) according to the experimental method described in theattached protocol. The results of the activity evaluation of variousdeletion clones thus obtained show that the region up to 4.9 kb isessential for expression of the activity and a region from 4.9 kb to 5.8kb is not especially required for degradation activity.

EXAMPLE 5 Sequencing of Toluene Monooxygenase Gene

[0346] The nucleotide sequence of pKK01 was determined as follows. pKK01was digested by various restriction enzymes and subcloned into pUC18plasmid. Deletion clones were prepared from pKK01 or subclones ofpartial pKK01 using Deletion Kit for Kilo-Sequence (Takara Shuzo Co.,Ltd.) to determine the nucleotide sequence of the 5.8-kb fragmentencoding toluene monooxygenase by the dideoxy method. The dideoxy methodwas carried out using ABI PRISM Cycle Sequencing Kit (Perkin ElmerCorporation) according to the attached protocol for reaction conditions,etc. DNA recombination and Kilo-Sequence method were also performedaccording to the conventional methods or the manufacturer's protocolsattached. The results of sequencing show that the DNA encoding toluenemonooxygenase is contained in 5,828 bases comprised of 7 coding regionsas shown by SEQ ID NO:1; a region tomK encoding the amino acid sequenceTomK of SEQ ID NO:2 ; a region TomL encoding the amino acid sequencetomL of SEQ ID NO:3; a region tomM encoding an amino acid sequence TomMof SEQ ID NO:4; a region tomN encoding an amino acid sequence TomN ofSEQ ID NO:5; a region tomO encoding an amino acid sequence (TomO) of SEQID NO:6; a region tomP encoding an amino acid sequence TomP of SEQ IDNO:7; and a region tomQ encoding an amino acid sequence TomQ of SEQ IDNO:8.

[0347] Here, considering the results of Example 4 together, thepolypeptide (TomK)(SEQ ID NO:2) encoded by tomK is not essential forexpression of the activity but the presence of TomK clearly enhances thetoluene monooxygenase activity. It is therefore desirable for sufficientexpression of the activity that TomK is present as a component oftoluene monooxygenase. The polypeptide (TomQ)(SEQ ID NO:8) encoded bytomQ is not essential for expression of the activity. In addition, thetoluene monooxygenase activity is not affected by the presence of TomQ.Thus, it is not essential to contain TomQ as a component of toluenemonooxygenase.

[0348] In other words, any DNA fragment containing segments encoding theamino acid sequences of SEQ ID NOs:3-7 as the components of toluenemonooxygenase where these segments are aligned so that expressed TomL toTomP having the amino acid sequences of SEQ ID NOs:3-7 can form aprotein with a toluene monooxygenase activity is included in thepreferred DNA fragment of the present invention. DNA fragments withvariation in at least one segment of the DNA fragment with the provisothat the activity of toluene monooxygenase is not impaired are includedin the preferred DNA fragments of the present invention.

[0349] DNA fragments further containing a region encoding the amino acidsequence TomK of SEQ ID NO:2 or a variant in which the amino acidsequence of SEQ ID NO:2 is changed with the proviso that it does notimpair the property to enhance a toluene monooxygenase activity are alsoincluded in the preferred embodiment of the present invention.

[0350] It should be noted that, in tomK, a sequence corresponding to SDsequence is not found before the 1st ATG (216-218) but present beforethe 2nd ATG (234-236). Thus, in the following Sequence Listing, thepolypeptide encoded by the nucleotide sequence beginning the base number234 is designated as TomK.

[0351] In addition, in tomL, a sequence corresponding to SD sequence isnot found before the 1st ATG (bases number 391-393) but present beforeGTG(463-465). Thus, in the following Sequence Listing, the polypeptideencoded by the nucleotide sequence beginning the base of number 463 isdesignated as SEQ ID: NO.3 (TomL).

EXAMPLE 6 Recombination of Toluene Monooxygenase Gene into ExpressionVectors

[0352] As expression vectors, pTrc99A (Amarsham-Pharmacia), pSE280(Invitrogen), and pSE380 (Invitrogen) were employed. They contain anampicillin-resistant gene as a marker, and pTrc99A has a sequencederived from pBR322, and pSE280 and pSE380 have those derived from ColE1as ori. All these 3 vectors contain a trc promoter and a rrnBterminator, and a ribosome-binding site is located before the NcoIrestriction site. lacIQ is contained in pTrc99A and pSE380 but not inpSE280.

[0353] To incorporate the toluene monooxygenase gene into these vectors,NcoI restriction sites were introduced in tomK and tomL. The following 5primers (Takara Shuzo Co., Ltd.) were prepared to introduce the NcoIrestriction site by PCR: SEQ ID NO: 9 tom-K15′-AGTCCGCCATGGAGGCGACACCGATCATGAATCAGC-3′ 36 mer SEQ ID NO: 10 tom-K25-CACCGACCATGGATCAGCACCCCACCGATCTTTC-3′ 34 mer SEQ ID NO: 11 tom-L15′-TGCCGCCTTCCATGGGTTCTGCCGCGAACAGCAG-3′ 34 mer SEQ ID NO: 12 tom-L25′-AGCAAGCCATGGCCATCGAGCTGAAGACAGTCGACATCA-3′ 39 mer SEQ ID NO: 13 tail5′-CCGACCATCACCTGCTCGGCCAGATGGAAGTCGAG-3′ 35 mer

[0354] The tom-K1 was designed to introduce the NcoI restriction site atthe 1st ATG region (bases 216-218 in the Sequence Listing) of tomK.Similarly, tom-K2 was designed to introduce the NcoI site at the 2nd ATGregion (bases 234-236 in the SEQ ID NO:1) of tomK; tomL-1 was designedto introduce the NcoI site at the 1st ATG region (bases 391-393 in SEQID NO:1) of tomL; and tom-L2 was designed to introduce the NcoI site atthe 1st GTG region (bases 463-465 in SEQ ID NO:1) of tomL. Using primercombinations of the primer (5) with the respective primers (1)-(4) andthe 8.5 kb fragment-containing plasmid DNA of FERM BP-6916 as thetemplate, PCR was performed. PCR was carried out using Takara LA PCR KitVer. 2 (Takara Shuzo Co., Ltd.) with a reaction volume of 50 μl,repeating 30 times a cycle of reaction at 94° C. for 1 minute and 98° C.for 20 seconds followed by 72° C. for 5 minutes (shuttle PCR), thenfollowed by reaction at 72° C. for 10 minutes. The reaction conditionswere according to the manufacturer's protocol.

[0355] As a result, the combinations of the primers (1) and (5), (2) and(5), (3) and (5), and (4) and (5) gave the PCR products of about 5.6 kb,about 5.6 kb, about 5.4 kb, and about 5.4 kb, respectively. Therespective DNA fragments were digested with the restriction enzyme NcoI(Takara Shuzo Co., Ltd.) to give the respective fragments of about 5.0kb, about 5.0 kb, about 4.9 kb, and about 4.8 kb together with afragment of about 0.6 kb. It shows that PCR products were completelydigested by the restriction enzyme NcoI. These NcoI-digested productswere purified using a spin column HR-4000 (Amarsham-Pharmacia) and usedfor the following ligation reaction.

[0356] The above expression vectors were completely digested with therestriction enzyme NcoI, dephosphorylated, subjected to phenoltreatment, and purified with a spin column HR-400 (Amarsham-Pharmacia).The vectors were then ligated to the NcoI-digested PCR products totransform E.coli HB101 (Takara Shuzo Co., Ltd.) according to theconventional method. The transformed E.coli HB101 cells were then grownon LB plate containing 100 μg/ml of ampicillin for transformantselection. After the transformants were cultured in LB medium at 37° C.overnight, plasmid DNA was extracted by the alkaline method to examinethe recombinant plasmids. Transformants in which the respective PCRfragments were accurately inserted into the NcoI restriction site of therespective expression vectors were obtained.

[0357] A list of the obtained recombinant plasmids are shown in Table 3.TABLE 3 tom-K1 tom-K2 tom-L1 tom-L2 pTrc99A pK19 pK29 pL19 pL29 pSE280pK12 pK22 pL12 pL22 pSE380 pK13 pK23 pL13 pL23

EXAMPLE 7 Ability of E.coli HB101 Recombinant Strains to DegradeAromatic Compounds and Chlorinated Aliphatic Hydrocarbon Compounds(without Induction with IPTG)

[0358] The cells of the E.coli strains, each harboring one of the 12recombinant plasmids obtained as described in Example 6, were inoculatedin 100 ml of LB medium, cultured at 37° C. overnight, harvested, washed,and suspended in an M9+mineral solution. Ten ml aliquots of thesuspension were placed in 27.5 ml vials, and each vial was tightlysealed with a Teflon-coated butyl rubber stopper and aluminium seal.Then, gaseous trichloroethylene (TCE), cis-1,2-dichloroethylene(cis-1,2-DCE), trans-1,2-dichloroethylene (trans-1,2-DCE),1,1-dichloroethylene (1,1-DCE), toluene, and benzene were added torespective vials with a syringe to a concentration of 20 ppm (supposingall of the introduced substance dissolved in the aqueous phase in thevial). The vials were shaken and incubated at 30° C. The concentrationsof the respective compounds in the gas phase after 6 hour incubationwere measured by gas chromatography. The results are shown in Table 4.E.coli HB101(pSE280) was employed as a control and degradation wasevaluated in the same manner. TABLE 4 pK19 pK29 pL19 pL29 pK12 pK22 pL12pL22 TCE 4.5 5.2 7.8 7.5 0 0 0.4 0.2 cis-1,2-DCE 2.5 2.4 3.8 4.5 0 0 2.13.2 trans-1,2- 3.1 4.2 5.2 5.8 0 0 1.5 1.4 DCE 1,1-DCE 7.2 6.6 8.9 9.1 00 1.2 0.9 Toluene 1.3 1.1 2.5 3.2 0 0 0 0 Benzene 4.8 5.1 7.3 6.8 0 00.9 0.5 pK13 pK23 pL13 pL23 pSE280 TCE 3.8 4.3 5.5 5.3 20.1 cis-1,2-DCE0.9 0.7 1.5 1.8 18.9 trans-1,2- 1.2 1.1 2.1 2.1 19.8 DCE 1,1-DCE 2.5 2.45.1 4.9 20.7 Toluene 1.2 0.9 1.8 1.7 21.0 Benzene 3.5 3.3 4.8 4.4 20.2

[0359] Similarly, phenol, ortho-cresol, meta-cresol and para-cresol wereintroduced into respective 27.5 ml vials each containing 10 ml of thecell suspension at a concentration of 50 ppm. Each vial was tightlysealed with a butyl rubber stopper and aluminum seal. The vials wereshaken and incubated at 30° C. The quantities of the respectivecompounds in the liquid phase were determined by the amino antipyrinemethod with a spectrophotometer to determine their concentrations after6 hours. The results are shown in Table 5. E.coli HB101(pSE280) wasemployed as a control and degradation was evaluated in the same manner.TABLE 5 pK19 pK29 pL19 pL29 pK12 pK22 pL12 pL22 Phenol 0 0 0 0 0 0 0 0Ortho-cresol 0 0 0 0 0 0 0 0 Meta-cresol 0 0 0 0 0 0 0 0 Para-cresol 0 00 0 0 0 0 0 pK13 pK23 pL13 pL23 pSE280 Phenol 0 0 0 0 50.6 Ortho-cresol0 0 0 0 52.5 Meta-cresol 0 0 0 0 53.1 Para-cresol 0 0 0 0 50.5

[0360] The above results confirm that E.coli HB101 transformantsharboring the expression vectors have an excellent ability to degradethe aromatic compounds and chlorinated aliphatic hydrocarbon compounds.It is shown that transformants harboring pTrc99A or pSE380-derivedexpression vectors express a lower degrading activity in a system notcontaining IPTG than those harboring pSE280-derived plasmids, sincepSE280 lacks lacIq.

EXAMPLE 8 Ability of E.coli HB101 Transformants harboring ExpressionVectors to Degrade Aromatic Compounds and Chlorinated AliphaticHydrocarbon Compounds (with Induction with IPTG)

[0361] Each E.coli HB101 transformant strain harboring one of the 12recombinant plasmids obtained as described in Example 6, was inoculatedin 100 ml of LB medium, cultured at 37° C. to reach OD₆₀₀ of about 0.8,and then IPTG was added to 1 mM concentration followed by furtherincubation at 37° C. for 5 hours. Then the cells were harvested, washedand suspended in an M9+ mineral solution. Ten ml aliquots of thesuspension were placed in 27.5 ml vials, and each vial was tightlysealed with a Teflon-coated butyl rubber stopper and aluminium seal.Then, gaseous trichloroethylene (TCE), cis-1,2-dichloroethylene(cis-1,2-DCE), trans-1,2-dichloroethylene (trans-1,2-DCE),1,1-dichloroethylene (1,1-DCE), toluene, and benzene were added torespective vials with a syringe to a concentration of 20 ppm (supposingall of the introduced substance dissolved in the aqueous phase in thevial). The vials were shaken and incubated at 30° C. The concentrationsof the respective compounds in the gas phase after 6 hour incubationwere measured by gas chromatography. The results are shown in Table 6.E.coli HB101(pSE280) was employed as a control and degradation wasevaluated in the same manner. TABLE 6 pK19 pK29 pL19 pL29 pK12 pK22 pL12pL22 TCE 0 0 0 0 0 0 0.7 0.5 cis-1,2-DCE 0 0 0 0 0 0 1.9 2.1 trans-1,2-0 0 0 0 0 0 0.9 1.9 DCE 1,1-DCE 0 0 0.7 0.5 0 0 0.8 0.7 Toluene 0 0 0 00 0 0 0 Benzene 0 0 1.2 2.1 0 0 1.3 0.9 pK13 pK23 pL13 pL23 pSE280 TCE 00 0 0 21.2 cis-1,2-DCE 0 0 0 0 19.9 trans-1,2- 0 0 0 0 20.7 DCE 1,1-DCE0 0 0 0 19.8 Toluene 0 0 0 0 20.5 Benzene 0 0 0.3 0.1 21.0

[0362] Similarly, phenol, ortho-cresol, meta-cresol and para-cresol wereintroduced into respective 27.5 ml vials each containing 10 ml of thecell suspension, at a concentration of 50 ppm. Each vial was tightlysealed with a butyl rubber stopper and aluminum seal. The vials wereshaken and incubated at 30° C. The quantities of the respectivecompounds in the liquid phase were determined by the amino antipyrinemethod with a spectrophotometer to determine their concentrations after6 hours. The results are shown in Table 7. E.coli HB101(pSE280) wasemployed as a control and degradation was evaluated in the same manner.TABLE 7 pK19 pK29 pL19 pL29 pK12 pK22 pL12 pL22 Phenol 0 0 0 0 0 0 0 0Ortho-cresol 0 0 0 0 0 0 0 0 Meta-cresol 0 0 0 0 0 0 0 0 Para-cresol 0 00 0 0 0 0 0 pK13 pK23 pL13 pL23 pSE280 Phenol 0 0 0 0 50.0 Ortho-cresol0 0 0 0 51.1 Meta-cresol 0 0 0 0 52.3 Para-cresol 0 0 0 0 47.9

[0363] The above results confirm that E.coli HB101 transformantsharboring toluene monooxygenase-expression vectors has an excellentability to degrade aromatic compounds and chlorinated aliphatichydrocarbon compounds. It is shown that transformants harboring pTrc99A-or pSE380-based expression vectors show more excellent degradingactivity by IPTG induction.

EXAMPLE 9 TCE Degradation by E.coli HB101(pK22) and HB101(pK23)recombinant Strains in Soil (Without IPTG Induction)

[0364]E.coli HB101(pK22) and HB101(pK23) recombinant strains asdescribed in Example 6 were respectively inoculated in 10 ml of LBmedium and cultured at 37° C. overnight. Fifty grams of Sawara sievedsand (unsterilized) was placed in 68 ml vials each. Five ml of LB mediuminoculated with the above seed culture to 100:1, was then added to thesand in each vial. Each vial was cotton-plugged, and incubated at 37° C.for 8 hours without shaking. After that, each vial was tightly sealedwith a Teflon-coated butyl rubber stopper and aluminum seal. Gaseous TCEwas introduced into the vials with a syringe to 20 ppm ( supposing allTCE dissolved into the aqueous phase in the vial). The vials wereincubated at 30° C. Quantitative analysis of TCE in the gas phase werecarried out by gas chromatography after 6 hours to determine TCEconcentrations. The results are shown in Table 8. E.coli HB101(pSE280)was employed as a control and evaluated in the same manner. TABLE 8 pK22pK23 pSE280 TCE 0 2.4 20.8

[0365] The above results confirm that E.coli HB101 transformantsharboring pK22 and pK23 also show an excellent TCE-degrading ability insoil. It is shown that transformant harboring pK23 (pSE380-based)expresses a lower degrading activity in a system not containing IPTGthan that harboring pSE280-derived plasmid pK23, since the formercontains lacIq.

EXAMPLE 10 TCE Degradation by E.coli HB101(pK22) or HB101(pK23) in Soil(With IPTG Induction)

[0366] The cells of E.coli HB101(pK22) and HB101(pK23) recombinantstrains as described in Example 6 were respectively inoculated in 10 mlof LB medium and cultured at 37° C. overnight. Fifty grams of Sawarasieved sand (unsterilized) were placed in 68 ml vials each. Five ml ofLB medium inoculated with the above seed culture to 100:1, was thenadded to the sand. Each vial was cotton-plugged, and incubated at 37° C.for 4 hours without shaking. Then 1 ml of a 10 mM IPTG solution wasadded to each vial. After that, each vial was tightly sealed with aTeflon-coated butyl rubber stopper and aluminum seal. Gaseous TCE wasintroduced into the vials with a syringe to 20 ppm ( supposing all TCEdissolved into the aqueous phase in the vial). The vials were incubatedat 30° C. Quantitative analysis of TCE in the gas phase were carried outby gas chromatography after 6 hours to determine TCE concentrations. Theresults are shown in Table 9. E.coli HB101(pSE280) was employed as acontrol and evaluated in the same manner. TABLE 9 pK22 pK23 pSE280 TCE 00 20.3

[0367] The above results confirm that E.coli HB101 transformantsharboring pK22 and pK23 also show an excellent TCE-degrading ability insoil. It is shown that transformant harboring pK23 (pSE380-based)expresses higher degrading activity with IPTG induction.

EXAMPLE 11 TCE Degradation by E.coli HB101(pK22) or HB101(pK23) in GasPhase (Without IPTG Induction)

[0368] The cells of respective recombinant strains, E.coli HB101(pK22)and HB101(pK23) as described in Example 6, were inoculated in 100 ml ofLB medium and cultured at 37° C. overnight. Aliquots (30 ml) of eachseed culture were transferred into 68 ml vials, into which air which hadpassed through a saturation TCE solution was introduced at a flow rateof 20 ml/min for 10 minutes. Each vial was tightly sealed with aTeflon-coated butyl rubber stopper and aluminum seal, and shakingculture was conducted at 30° C. Quantitative analysis of TCE in the gasphase were carried out by gas chromatography to determine itsconcentration after 6 hours. The results are shown in Table 10. E.coliHB101(pSE280) was employed as a control and degradation was evaluated inthe same manner. TABLE 10 pK22 pK23 pSE280 TCE 0 12.1 47.9

[0369] The above results confirm that recombinant E.coli HB101(pK22) orHB101(pK23) shows an excellent TCE-degrading ability also in the gasphase. It is shown that transformant harboring pK23 (pSE380-based)expresses a lower degrading activity in a system not containing IPTGthan that harboring pSE280-derived plasmid pK23, since the formercontains lacIq.

EXAMPLE 12 TCE Degradation by recombinant E.coli HB101(pK22) andHB101(pK23) in Gas Phase (With IPTG Induction)

[0370]E.coli (HB101) recombinant strains each harboring pK22 or pK23 asdescribed in Example 6 were respectively inoculated into 100 ml of LBmedium and cultured at 37° C. to reach OD₆₀₀ of about 0.8, and then IPTGwas added to 1 mM concentration followed by further incubation at 37° C.for 5 hours. Aliquots (30 ml) of the cell suspension were transferredinto 68 ml vials, into which air which had passed through in a saturatedTCE solution was introduced at a flow rate of 20 ml/min for 10 minutes.Each vial was tightly sealed with a Teflon-coated butyl rubber stopperand aluminum seal, and shaking culture was conducted at 30° C.Quantitative analysis of TCE in the gas phase were carried out by gaschromatography to determine its concentration after 6 hours. The resultsare shown in Table 11. E.coli HB101(pSE280) was employed as a controland degradation was evaluated in the same manner. TABLE 11 pK22 pK23pSE280 TCE 0 0 54.2

[0371] The above results confirm that recombinant E.coli HB101(pK22) orHB101(pK23) shows an excellent TCE-degrading ability also in the gasphase, and show that transformant harboring pK23 (pSE380-based)expresses higher degrading activity with IPTG induction.

EXAMPLE 13 Introduction of Recombinant Plasmid containing TolueneMonooxygenase Gene into Vibrio sp. strain KB1

[0372] The toluene monooxygenase gene beginning from the second ATG oftomK (base number 234-236) was transferred from the recombinant plasmidpK29 of Example 6 (recombinant pTrc99A containing the gene) into avector pBBR122 (Mo Bi Tec) having a wide host range replication regionnot belonging to an incompatible group of IncP, IncQ, and IncW. Thisrecombinant plasmid was introduced in Vibrio sp. strain KB1, and itsability to degrade aromatic compounds and chlorinated aliphatichydrocarbon compounds was evaluated.

[0373] First, a wide host range recombinant plasmid was constructed. Anabout 7.0-kb fragment containing the toluene monooxygenase gene, a trcpromoter, and a rrnB terminator was cut out from pK29 using therestriction enzymes HpaI (Takara Shuzo Co., Ltd.) and SmaI (Takara ShuzoCo., Ltd.). This fragment of about 7.0 kb does not contain the lacIqsequence. As a vector of a wide host range, pBBR122 was employed.pBBR122 was completely digested with the restriction enzyme SmaI (TakaraShuzo Co., Ltd.). The 7.0 kb fragment containing the toluenemonooxygenase gene, a trc promoter, and an rrnB terminator prepared asdescribed above was ligated to the SmaI restriction site of the pBBR122using DNA Ligation Kit Ver. 2 (Takara Shuzo Co., Ltd.) and therecombinant plasmid thus constructed was introduced into E.coli HB101(Takara Shuzo Co., Ltd.). The cells of the E.coli thus treated wereapplied on LB plate containing 50 μg/ml of chloramphenicol as aselection agent. When the colonies on the plate grew to an appropriatesize, the colonies were transferred by replica printing onto an LB platecontaining 50 μg/ml of kanamycin as a selection agent. Transformantsthat could proliferate on the plate with chloramphenicol but not on theplate with kanamycin were selected, and cultured in LB medium at 37° C.overnight, to extract plasmid DNA from the cells by the alkaline method.After checking the plasmids, transformants harboring a recombinantplasmid where the 7.0 kb fragment was correctly inserted into the SmaIsite of the pBBR122 were obtained. The recombinant plasmid thus obtainedwas about 12.3 kb in length and designated as pK29bbr.

[0374] The SOB medium shown below was employed for liquid culture ofVibrio sp. strain KB1. Chloramphenicol was used at a concentration of 50μg/ml as a selection agent and the culture temperature was 30° C. Therecombinant plasmid pK29 was introduced into Vibrio sp. strain KB1 cellsby electroporation using a gene pulsar (Bio-Rad). The recombinantplasmid pK29bbr was stably retained after introduction into Vibrio sp.strain KB1.

[0375] SOB medium:

[0376] Trypton: 20 g

[0377] Yeast extract: 5 g

[0378] NaCl: 0.5 g

[0379] 250 mM KCl: 10 ml

[0380] Distilled water (to 990 ml)

[0381] pH 7.0

[0382] The above solution was sterilized by autoclaving and cooled toroom temperature, to which 10 ml of a 2 M Mg solution (1 MMgSO_(4.7)H₂O+1 M MgCl_(2.6)H₂O) separately sterilized by autoclavingwas added.

EXAMPLE 14 Ability of Vibrio sp. KB1(pK29bbr) to Degrade AromaticCompounds and Chlorinated Aliphatic Hydrocarbon Compounds

[0383] The cells of Vibrio sp. KB1(pK29bbr) were inoculated in 100 ml ofSOB medium, cultured at 30° C. overnight, harvested, washed, and thensuspended in 100 ml of M9 (containing 6.2 g of Na₂HPO₄, 3.0 g of KH₂PO₄,0.5 g of NaCl, and 1.0 g of NH₄Cl per liter) supplemented with a mineralstock solution (3 ml to 1 liter of M9 medium).

[0384] Ten ml of the suspension was placed in respective 27.5 ml vialsand each vial was tightly sealed with a Teflon-coated butyl rubberstopper and aluminum seal. Then, gaseous trichloroethylene (TCE),cis-1,2-dichloroethylene (cis-1,2-DCE), trans-1,2-dichloroethylene(trans-1,2-DCE), 1,1-dichloroethylene (1,1-DCE), toluene, and benzenewere added to respective vials with a syringe to a concentration of 20ppm (supposing all of the introduced substance dissolved in the aqueousphase in the vial). The vials were shaken and incubated at 30° C. Theconcentrations of the respective compounds in the gas phase after 6 hourincubation were measured by gas chromatography. The results are shown inTable 12. Vibrio sp. KB1(pBBR122) was tested as a control anddegradation was evaluated in the same manner. TABLE 12 KB1 (pK29bbr) KB1(pBBR122) TCE 0 19.1 cis-1,2-DCE 0 20.2 trans-1,2-DCE 0 21.3 1-1,DCE 019.2 Toluene 0 19.8 Benzene 0 21.0

[0385] Similarly, to 10 ml of the prepared cell suspension in a 27.5-mlvial, phenol, ortho-cresol, meta-cresol, and para-cresol were added to50 ppm, respectively. The vial was tightly sealed with a butyl rubberstopper and aluminum seal, and then shaken and incubated at 30° C. Thequantities of the respective compounds in the liquid phase were measuredby the amino antipyrine method with a spectrophotometer to obtain theirconcentrations after 6 hours. The results are shown in Table 13. Vibriospecies strain KB1 containing only pBBR122 was employed as a control anddegradation was evaluated in a similar system.

[0386] Similarly, phenol, ortho-cresol, meta-cresol and para-cresol wereintroduced at a concentration of 50 ppm into respective 27.5 ml vialseach containing 10 ml of the cell suspension. Each vial was tightlysealed with a butyl rubber stopper and aluminum seal, and shaken andincubated at 30° C. The quantities of the respective compounds in theliquid phase were determined by the amino antipyrine method using aspectrophotometer to determine their concentrations after 6 hours. Theresults are shown in Table 13. Vibrio sp. KB1(pBBR122) was tested as acontrol and degradation was evaluated in the same manner. TABLE 13 KB1(pK29bbr) KB1 (pBBR122) Phenol 0 51 Ortho-cresol 0 50 Meta-cresol 0 49Para-cresol 0 50

[0387] The above results show that the recombinant Vibrio sp. strain KB1harboring pK29bbr can constitutively express the ability to degradearomatic compounds and chlorinated aliphatic hydrocarbon compounds.

EXAMPLE 15 Degradation of TCE by Recombinant Vibrio sp. KB1(pK29bbr) inSoil

[0388] Vibrio sp. KB1(pK29bbr) recombinant strain as described inExample 13 was inoculated in 10 ml of SOB medium and cultured at 30° C.overnight. Fifty grams of Sawara sieved sand (unsterilized) was placedin each 68 ml vial. Five ml of SOB medium inoculated with the above seedculture to 100:1 was then added to the sand in each vial. Each vial wascotton-plugged and incubated at 30° C. for 12 hours without shaking.After that, each vial was tightly sealed with a Teflon-coated butylrubber stopper and aluminum seal. Gaseous TCE was introduced into thevials with a syringe to 20 ppm ( supposing all TCE dissolved into theaqueous phase in the vial). The vials were incubated at 30° C.Quantitative analysis of TCE in the gas phase were carried out by gaschromatography after 6 hours to determine TCE concentrations. Theresults are shown in Table 14. Vibrio sp. KB1(pBBR122) was tested as acontrol and degradation was evaluated in the same manner. TABLE 14 KB1(pK29bbr) KB1 (pBBR122) TCE 0 20.2

[0389] The above results show that the recombinant Vibrio sp.KB1(pK29bbr) can constitutively express the ability to degrade TCE alsoin soil.

EXAMPLE 16 Degradation of TCE by Recombinant Vibrio sp. KB1(pK29bbr) inGas Phase

[0390] The cells of recombinant Vibrio sp. KB1(pK29bbr) as described inExample 13 were inoculated in 100 ml of SOB medium and cultured at 30°C. overnight. Aliquots (30 ml) of the seed culture were transferred into68 ml vials, into which air which had passed through a saturation TCEsolution was introduced at a flow rate of 20 ml/min for 10 minutes. Eachvial was tightly sealed with a Teflon-coated butyl rubber stopper andaluminum seal, and shaking culture was conducted at 30° C. Quantitativeanalysis of TCE in the gas phase were carried out by gas chromatographyto determine its concentration after 6 hours. The results are shown inTable 15. Vibrio sp. KB1(pBBR122) was employed as a control anddegradation was evaluated in the same manner. TABLE 15 KB1 (pK29bbr) KB1(pBBR122) TCE 0 52.1

[0391] The above results show that the recombinant Vibrio sp.KB1(pK29bbr) can constitutively express the ability to degrade TCE alsoin the gas phase.

[0392] According to the present invention, a DNA fragment carrying atoluene monooxygenase gene with an excellent ability to degrade aromaticcompounds and chlorinated aliphatic hydrocarbon compounds can beobtained. In addition, a novel recombinant plasmid containing the DNAfragment as a whole or a part thereof that can be utilized to obtain atransformant capable of degrading aromatic compounds and/or chlorinatedaliphatic hydrocarbon compounds can be obtained. Further, a transformantharboring the plasmid and can be utilized to degrade aromatic compoundsand/or chlorinated aliphatic hydrocarbon compounds can be obtained.Furthermore, a practical method for environmental remediation that canefficiently degrade either aromatic compounds and/or chlorinatedaliphatic hydrocarbon compounds by utilizing the transformant.

1 13 1 5828 DNA Burkholderia cepacia CDS (234)..(443) tomK 1 gatcatttcatcaaatgcgc tcgagcgggt tgctcaaatg atgaaaaagg ccaccggaca 60 tgggtttcggcacgatcgcc ggcgggcgtt ttccgttctg gttaaccgcc attgtgggtc 120 gcgaaatttaacttcgcgtc agggctttcc ctgaattatc gagatttttt gctgcctggg 180 tcgaacgtggcacggatgct gcattgaagt ccggcatgga ggcgacaccg atc atg 236 Met 1 aat cagcac ccc acc gat ctt tcc ccg ttc gat ccc ggc cgc aag tgc 284 Asn Gln HisPro Thr Asp Leu Ser Pro Phe Asp Pro Gly Arg Lys Cys 5 10 15 gtc cgc gtgacc ggc acg aac gcg cgc ggc ttc gtc gaa ttc gag ctg 332 Val Arg Val ThrGly Thr Asn Ala Arg Gly Phe Val Glu Phe Glu Leu 20 25 30 tcg atc ggc ggcgcg ccg gaa ctg tgc gtc gag ctg acg ttg tct cct 380 Ser Ile Gly Gly AlaPro Glu Leu Cys Val Glu Leu Thr Leu Ser Pro 35 40 45 gcc gcc ttc gat gcgttc tgc cgc gaa cag cag gtc acg cgg ctc gac 428 Ala Ala Phe Asp Ala PheCys Arg Glu Gln Gln Val Thr Arg Leu Asp 50 55 60 65 gtc gaa gcg aac ccatgaccttgag gagcaagaa gtg acc atc gag ctg aag 480 Val Glu Ala Asn Pro MetThr Ile Glu Leu Lys 70 75 aca gtc gac atc aag ccg ctc cgg cac acc tttgcg cat gtc gcg cag 528 Thr Val Asp Ile Lys Pro Leu Arg His Thr Phe AlaHis Val Ala Gln 80 85 90 aac atc ggc ggc gac aag acg gcg acg cgc tac caggaa ggc atg atg 576 Asn Ile Gly Gly Asp Lys Thr Ala Thr Arg Tyr Gln GluGly Met Met 95 100 105 ggc gcg cag ccc cag gag aac ttc cat tac cgg ccgacc tgg gac ccg 624 Gly Ala Gln Pro Gln Glu Asn Phe His Tyr Arg Pro ThrTrp Asp Pro 110 115 120 gac tac gag atc ttc gat ccg tcg cgc tcg gcg atccgg atg gcg aac 672 Asp Tyr Glu Ile Phe Asp Pro Ser Arg Ser Ala Ile ArgMet Ala Asn 125 130 135 140 tgg tac gcg ttg aag gat ccg cgc cag ttc tactac gcg tcg tgg gcg 720 Trp Tyr Ala Leu Lys Asp Pro Arg Gln Phe Tyr TyrAla Ser Trp Ala 145 150 155 acc acg cgg gcg cgc cag cag gat gcg atg gagtcg aac ttc gag ttc 768 Thr Thr Arg Ala Arg Gln Gln Asp Ala Met Glu SerAsn Phe Glu Phe 160 165 170 gtc gaa tcg cgc cgg atg atc ggc ctg atg cgcgac gac gtg gcc gcg 816 Val Glu Ser Arg Arg Met Ile Gly Leu Met Arg AspAsp Val Ala Ala 175 180 185 cgg gcg ctc gac gtg ctg gtg ccg ctg cgc cacgcc gcg tgg ggc gcg 864 Arg Ala Leu Asp Val Leu Val Pro Leu Arg His AlaAla Trp Gly Ala 190 195 200 aac atg aac aac gcg cag atc tgc gcg ctc ggctac ggc acg gtg ttc 912 Asn Met Asn Asn Ala Gln Ile Cys Ala Leu Gly TyrGly Thr Val Phe 205 210 215 220 acc gcg ccc gcg atg ttc cat gcg atg gacaac ctc ggc gtc gcg caa 960 Thr Ala Pro Ala Met Phe His Ala Met Asp AsnLeu Gly Val Ala Gln 225 230 235 tac ctc acg cgt ctc gcg ctc gcg atg gccgag ccc gac gtg ctg gag 1008 Tyr Leu Thr Arg Leu Ala Leu Ala Met Ala GluPro Asp Val Leu Glu 240 245 250 gcg gcc aag gcg acc tgg acc cgc gac gccgcc tgg cag ccg ctg cgc 1056 Ala Ala Lys Ala Thr Trp Thr Arg Asp Ala AlaTrp Gln Pro Leu Arg 255 260 265 cgc tac gtc gag gac acg ctg gtc gtc gccgat ccg gtc gag ctg ttc 1104 Arg Tyr Val Glu Asp Thr Leu Val Val Ala AspPro Val Glu Leu Phe 270 275 280 atc gcg cag aac ctc gcg ctc gac ggc ctgctg tat ccg ctc gtc tac 1152 Ile Ala Gln Asn Leu Ala Leu Asp Gly Leu LeuTyr Pro Leu Val Tyr 285 290 295 300 gac cgc ttc gtc gac gaa cgg atc gcgctc gaa ggc ggc tcg gca gtc 1200 Asp Arg Phe Val Asp Glu Arg Ile Ala LeuGlu Gly Gly Ser Ala Val 305 310 315 gcg atg ctg acc gcg ttc atg ccc gaatgg cac acc gag tcg aac cgc 1248 Ala Met Leu Thr Ala Phe Met Pro Glu TrpHis Thr Glu Ser Asn Arg 320 325 330 tgg atc gac gcg gtc gtg aag acg atggcc gcc gaa tcc gac gac aac 1296 Trp Ile Asp Ala Val Val Lys Thr Met AlaAla Glu Ser Asp Asp Asn 335 340 345 cgc gcg ctg ctc gcc cgc tgg aca cgcgac tgg tcc gcg cgc gcc gag 1344 Arg Ala Leu Leu Ala Arg Trp Thr Arg AspTrp Ser Ala Arg Ala Glu 350 355 360 gcg gca ctg gca ccg gtg gcg gca cgcgcg ctg cag gat gcc ggg cgc 1392 Ala Ala Leu Ala Pro Val Ala Ala Arg AlaLeu Gln Asp Ala Gly Arg 365 370 375 380 gcg gcg ctc gac gaa gtg cgc gagcag ttc cac gca cgc gcg gcc agg 1440 Ala Ala Leu Asp Glu Val Arg Glu GlnPhe His Ala Arg Ala Ala Arg 385 390 395 ctc ggc atc gcg ctc tgacgacgggaatcctccct taacccaagg aatgccagc 1494 Leu Gly Ile Ala Leu 400 atg tcc aacgta ttc atc gcc ttt cag gcc aat gag gac tcc aga ccg 1542 Met Ser Asn ValPhe Ile Ala Phe Gln Ala Asn Glu Asp Ser Arg Pro 405 410 415 atc gtg gatgcg atc gtc gcc gac aac ccg cgc gcg gtg gtg gtc gag 1590 Ile Val Asp AlaIle Val Ala Asp Asn Pro Arg Ala Val Val Val Glu 420 425 430 tcg ccc ggcatg gtc aag atc gac gcg ccg gac cgg ctg acg atc cgc 1638 Ser Pro Gly MetVal Lys Ile Asp Ala Pro Asp Arg Leu Thr Ile Arg 435 440 445 cgc gaa acgatc gag gaa ctg acc ggc acg cgc ttc gac ctg cag cag 1686 Arg Glu Thr IleGlu Glu Leu Thr Gly Thr Arg Phe Asp Leu Gln Gln 450 455 460 465 ctc caggtc aac ctg atc acg ctg tca ggc cac atc gac gag gac gac 1734 Leu Gln ValAsn Leu Ile Thr Leu Ser Gly His Ile Asp Glu Asp Asp 470 475 480 gac gagttc acg ctg agc tgg tcg cac tgaacgccgc gccacgcgca 1781 Asp Glu Phe ThrLeu Ser Trp Ser His 485 490 ccgacaacac cggagacacg a atg gac acg cca acgctc aag aaa aaa ctc 1832 Met Asp Thr Pro Thr Leu Lys Lys Lys Leu 495 500ggc ctg aag gac cgc tac gcg gca atg acg cgc ggc ctc ggc tgg gag 1880 GlyLeu Lys Asp Arg Tyr Ala Ala Met Thr Arg Gly Leu Gly Trp Glu 505 510 515acg acc tac cag ccg atg gac aag gtc ttc ccg tac gac cgc tac gag 1928 ThrThr Tyr Gln Pro Met Asp Lys Val Phe Pro Tyr Asp Arg Tyr Glu 520 525 530ggc atc aag atc cac gac tgg gac aag tgg gtc gac ccg ttc cgc ctg 1976 GlyIle Lys Ile His Asp Trp Asp Lys Trp Val Asp Pro Phe Arg Leu 535 540 545acg atg gat gcg tac tgg aaa tac cag ggc gag aag gaa aag aag ctg 2024 ThrMet Asp Ala Tyr Trp Lys Tyr Gln Gly Glu Lys Glu Lys Lys Leu 550 555 560tac gcg gtg atc gac gcg ttc acg cag aac aac gcg ttc ctc ggc gtg 2072 TyrAla Val Ile Asp Ala Phe Thr Gln Asn Asn Ala Phe Leu Gly Val 565 570 575580 agc gac gcc cgc tac atc aac gcg ctg aag ctg ttc ctc cag ggc gtg 2120Ser Asp Ala Arg Tyr Ile Asn Ala Leu Lys Leu Phe Leu Gln Gly Val 585 590595 acg ccg ctc gaa tac ctc gcg cac cgc ggc ttc gcg cat gtc ggc cgg 2168Thr Pro Leu Glu Tyr Leu Ala His Arg Gly Phe Ala His Val Gly Arg 600 605610 cac ttc acc ggc gag ggc gcg cgc atc gcg tgc cag atg cag tcg atc 2216His Phe Thr Gly Glu Gly Ala Arg Ile Ala Cys Gln Met Gln Ser Ile 615 620625 gac gag ctg cgg cac tac cag acc gaa acg cat gcg atg tcg acg tac 2264Asp Glu Leu Arg His Tyr Gln Thr Glu Thr His Ala Met Ser Thr Tyr 630 635640 aac aag ttc ttc aac ggg ttc cat cac tcg aac cag tgg ttc gac cgc 2312Asn Lys Phe Phe Asn Gly Phe His His Ser Asn Gln Trp Phe Asp Arg 645 650655 660 gtg tgg tac ctg tcg gtg ccg aag tcg ttc ttc gag gac gcg tat tcg2360 Val Trp Tyr Leu Ser Val Pro Lys Ser Phe Phe Glu Asp Ala Tyr Ser 665670 675 tcg ggg ccg ttc gag ttc ctg acc gcg gtc agc ttc tcg ttc gaa tac2408 Ser Gly Pro Phe Glu Phe Leu Thr Ala Val Ser Phe Ser Phe Glu Tyr 680685 690 gtg ctg acg aac ctg ctg ttc gtg ccg ttc atg tcg ggc gcc gcc tac2456 Val Leu Thr Asn Leu Leu Phe Val Pro Phe Met Ser Gly Ala Ala Tyr 695700 705 aac ggt gac atg tcg acc gtc acg ttc ggc ttc tcc gcg cag tcg gac2504 Asn Gly Asp Met Ser Thr Val Thr Phe Gly Phe Ser Ala Gln Ser Asp 710715 720 gaa tcg cgt cac atg acg ctc ggc atc gaa tgc atc aag ttc ctg ctc2552 Glu Ser Arg His Met Thr Leu Gly Ile Glu Cys Ile Lys Phe Leu Leu 725730 735 740 gaa cag gac ccg gac aac gtg ccg atc gtg cag cgc tgg atc gacaag 2600 Glu Gln Asp Pro Asp Asn Val Pro Ile Val Gln Arg Trp Ile Asp Lys745 750 755 tgg ttc tgg cgc ggc tac cgg ctg ctg acg ctg gtc gcg atg atgatg 2648 Trp Phe Trp Arg Gly Tyr Arg Leu Leu Thr Leu Val Ala Met Met Met760 765 770 gac tac atg cag ccc aag cgc gtg atg agc tgg cgc gag tcg tgggag 2696 Asp Tyr Met Gln Pro Lys Arg Val Met Ser Trp Arg Glu Ser Trp Glu775 780 785 atg tac gcc gag cag aac ggc ggc gcg ctg ttc aag gat ctc gcgcgc 2744 Met Tyr Ala Glu Gln Asn Gly Gly Ala Leu Phe Lys Asp Leu Ala Arg790 795 800 tac ggc att cgc gag ccg aag ggc tgg cag gac gcc tgc gaa ggcaag 2792 Tyr Gly Ile Arg Glu Pro Lys Gly Trp Gln Asp Ala Cys Glu Gly Lys805 810 815 820 gat cac atc agc cac cag gcg tgg tcg acg ttc tac ggc ttcaac gcg 2840 Asp His Ile Ser His Gln Ala Trp Ser Thr Phe Tyr Gly Phe AsnAla 825 830 835 gcc tcg gcg ttc cac acc tgg gtg ccg acc gaa gac gaa atgggc tgg 2888 Ala Ser Ala Phe His Thr Trp Val Pro Thr Glu Asp Glu Met GlyTrp 840 845 850 ctg tcg gcg aag tat ccc gac tcg ttc gac cgc tac tac cgcccg cgc 2936 Leu Ser Ala Lys Tyr Pro Asp Ser Phe Asp Arg Tyr Tyr Arg ProArg 855 860 865 ttc gat cac tgg ggc gag cag gcc agg gcc ggc aac cgc ttctac atg 2984 Phe Asp His Trp Gly Glu Gln Ala Arg Ala Gly Asn Arg Phe TyrMet 870 875 880 aag acg ctg ccg atg ctg tgc cag acg tgc cag atc ccg atgctg ttc 3032 Lys Thr Leu Pro Met Leu Cys Gln Thr Cys Gln Ile Pro Met LeuPhe 885 890 895 900 acc gag ccg ggc aac ccg acg aag atc ggc gcg cgc gaatcg aac tac 3080 Thr Glu Pro Gly Asn Pro Thr Lys Ile Gly Ala Arg Glu SerAsn Tyr 905 910 915 ctc ggc aac aag ttc cac ttc tgc agc gac cac tgc aaggac atc ttc 3128 Leu Gly Asn Lys Phe His Phe Cys Ser Asp His Cys Lys AspIle Phe 920 925 930 gat cac gag ccg cag aaa tac gtg cag gcg tgg ctg ccggtg cac cag 3176 Asp His Glu Pro Gln Lys Tyr Val Gln Ala Trp Leu Pro ValHis Gln 935 940 945 atc cat cag ggc aac tgc ttc ccg ccc gat gcg gac ccgggc gcg gag 3224 Ile His Gln Gly Asn Cys Phe Pro Pro Asp Ala Asp Pro GlyAla Glu 950 955 960 ggc ttc gat ccg ctc gcc gcg gtg ctc gac tac tac gcggtg acg atg 3272 Gly Phe Asp Pro Leu Ala Ala Val Leu Asp Tyr Tyr Ala ValThr Met 965 970 975 980 ggc cgc gac aac ctc gat ttc gac ggc tcg gaa gaccag aag aac ttc 3320 Gly Arg Asp Asn Leu Asp Phe Asp Gly Ser Glu Asp GlnLys Asn Phe 985 990 995 gcg gcg tgg cgc ggc cag gcc acg cgc aactgacccgcaa cgacaagcaa 3370 Ala Ala Trp Arg Gly Gln Ala Thr Arg Asn 10001005 tcttgacgag ggcccgcgaa gcgccgatgc gcgaacgcgg gccgacagga gacaaac 3427atg gcc gtc atc gcg ctc aaa ccc tac gac ttc ccg gtg aag gat gcc 3475 MetAla Val Ile Ala Leu Lys Pro Tyr Asp Phe Pro Val Lys Asp Ala 1010 10151020 gtc gag aag ttt ccg gcg ccg ctg ctc tac gtg tgc tgg gaa aac cat3523 Val Glu Lys Phe Pro Ala Pro Leu Leu Tyr Val Cys Trp Glu Asn His1025 1030 1035 ctg atg ttc ccg gcg ccg ttc tgc ctg ccg ctg ccg ccc gacatg ccg 3571 Leu Met Phe Pro Ala Pro Phe Cys Leu Pro Leu Pro Pro Asp MetPro 1040 1045 1050 ttc ggc gcg ctg gcc ggc gac gtg ctg ccg ccc gtc tacggc tat cac 3619 Phe Gly Ala Leu Ala Gly Asp Val Leu Pro Pro Val Tyr GlyTyr His 1055 1060 1065 1070 ccc gac ttc gcg aag atc gac tgg gat cgc gtcgag tgg ttc cgg tcg 3667 Pro Asp Phe Ala Lys Ile Asp Trp Asp Arg Val GluTrp Phe Arg Ser 1075 1080 1085 ggc gag ccg tgg gcg ccg gac ccg gcg aagagc ctg gcc ggc aac ggc 3715 Gly Glu Pro Trp Ala Pro Asp Pro Ala Lys SerLeu Ala Gly Asn Gly 1090 1095 1100 ctc ggg cac aag gac ctg atc agc ttccgc acg ccc ggc ctc gac ggc 3763 Leu Gly His Lys Asp Leu Ile Ser Phe ArgThr Pro Gly Leu Asp Gly 1105 1110 1115 ctc ggc ggc gcg agc ttctgaccgccac gcggacgagc gaaccatc atg agc 3815 Leu Gly Gly Ala Ser Phe MetSer 1120 1125 cac caa ctt acc atc gag ccg ctg ggc gtc acg atc gag gtcgag gaa 3863 His Gln Leu Thr Ile Glu Pro Leu Gly Val Thr Ile Glu Val GluGlu 1130 1135 1140 gga cag acg atg ctc gat gcc gcg ctg cgc cag ggc atctac att ccg 3911 Gly Gln Thr Met Leu Asp Ala Ala Leu Arg Gln Gly Ile TyrIle Pro 1145 1150 1155 cac gcg tgc tgt cac ggg ctg tgc ggc acc tgc aaggtc gcc gtg ctc 3959 His Ala Cys Cys His Gly Leu Cys Gly Thr Cys Lys ValAla Val Leu 1160 1165 1170 gac ggc gag acc gat ccc ggc gat gcg aac ccgttc gcg ctg atg gat 4007 Asp Gly Glu Thr Asp Pro Gly Asp Ala Asn Pro PheAla Leu Met Asp 1175 1180 1185 1190 ttc gag cgc gag gaa ggc aag gcg ctcgcg tgc tgc gcg acg ctg cag 4055 Phe Glu Arg Glu Glu Gly Lys Ala Leu AlaCys Cys Ala Thr Leu Gln 1195 1200 1205 gcc gac acc gtg atc gag gcc gacgtc gac gag gag ccg gat gcg gaa 4103 Ala Asp Thr Val Ile Glu Ala Asp ValAsp Glu Glu Pro Asp Ala Glu 1210 1215 1220 atc atc ccg gtc agg gac ttcgcg gcc gac gtc acg cgc atc gaa cag 4151 Ile Ile Pro Val Arg Asp Phe AlaAla Asp Val Thr Arg Ile Glu Gln 1225 1230 1235 ctc acg ccg acc atc aagtcg atc cgc ctg aag ctg tcg cag ccg atc 4199 Leu Thr Pro Thr Ile Lys SerIle Arg Leu Lys Leu Ser Gln Pro Ile 1240 1245 1250 cgc ttc cag gcg ggccag tac gtg cag ctc gag att ccc ggc ctc ggg 4247 Arg Phe Gln Ala Gly GlnTyr Val Gln Leu Glu Ile Pro Gly Leu Gly 1255 1260 1265 1270 cag agc cgcgcg ttc tcg atc gcg aac gcg ccg gcc gac gtc gcg gcc 4295 Gln Ser Arg AlaPhe Ser Ile Ala Asn Ala Pro Ala Asp Val Ala Ala 1275 1280 1285 acc ggcgag atc gaa ctg aac gtg cgg cag gtg ccg ggc ggg ctc ggc 4343 Thr Gly GluIle Glu Leu Asn Val Arg Gln Val Pro Gly Gly Leu Gly 1290 1295 1300 acgggc tac ctg cac gag caa ctg gcg acg ggc gag cgc gtg cgc ctg 4391 Thr GlyTyr Leu His Glu Gln Leu Ala Thr Gly Glu Arg Val Arg Leu 1305 1310 1315tcg ggc ccg tac ggc cgc ttc ttc gtg cgt cgc tcg gcc gcg cgg ccg 4439 SerGly Pro Tyr Gly Arg Phe Phe Val Arg Arg Ser Ala Ala Arg Pro 1320 13251330 atg atc ttc atg gcc ggc ggg tcg ggg ctg tcg agc ccg cgc tcg atg4487 Met Ile Phe Met Ala Gly Gly Ser Gly Leu Ser Ser Pro Arg Ser Met1335 1340 1345 1350 atc gcg gac ctg ctc gca agc ggc gtc acc gcg ccg atcacg ctg gtc 4535 Ile Ala Asp Leu Leu Ala Ser Gly Val Thr Ala Pro Ile ThrLeu Val 1355 1360 1365 tac ggt cag cgc agc gcg cag gag ctc tac tac cacgac gaa ttc cgc 4583 Tyr Gly Gln Arg Ser Ala Gln Glu Leu Tyr Tyr His AspGlu Phe Arg 1370 1375 1380 gcg ctg gcc gaa cgc cat ccg aac ttc acg tacgtg ccg gcg ctg tcc 4631 Ala Leu Ala Glu Arg His Pro Asn Phe Thr Tyr ValPro Ala Leu Ser 1385 1390 1395 gaa ggc gca ccg cac gcg ggc ggc gac gtcgcg caa ggg ttc gtg cac 4679 Glu Gly Ala Pro His Ala Gly Gly Asp Val AlaGln Gly Phe Val His 1400 1405 1410 gac gtc gcg aag gca cat ttc ggc ggcgac ttc tcc ggg cac cag gcg 4727 Asp Val Ala Lys Ala His Phe Gly Gly AspPhe Ser Gly His Gln Ala 1415 1420 1425 1430 tac ctg tgc ggg ccg ccc gcgatg atc gac gcg tgc atc acg acg ctg 4775 Tyr Leu Cys Gly Pro Pro Ala MetIle Asp Ala Cys Ile Thr Thr Leu 1435 1440 1445 atg cag ggg cgc ctg ttcgag cgc gac atc tat cac gag aag ttc atc 4823 Met Gln Gly Arg Leu Phe GluArg Asp Ile Tyr His Glu Lys Phe Ile 1450 1455 1460 tcg gcg gcc gac gcgcaa cag acg cgc agc ccg ctg ttc cgg cgg gtg 4871 Ser Ala Ala Asp Ala GlnGln Thr Arg Ser Pro Leu Phe Arg Arg Val 1465 1470 1475 tgac atg gac gcgggc cgc gta tgc ggg acg gtc acg atc gcg cag acc 4920 Met Asp Ala Gly ArgVal Cys Gly Thr Val Thr Ile Ala Gln Thr 1480 1485 1490 gac gag cgc tatgcg tgc gtg tcc ggc gag tcg ctg ctg gcc ggc atg 4968 Asp Glu Arg Tyr AlaCys Val Ser Gly Glu Ser Leu Leu Ala Gly Met 1495 1500 1505 gcg aaa ctcggc cgg cgc ggc att ccg gtc ggc tgc ctg aac ggc ggg 5016 Ala Lys Leu GlyArg Arg Gly Ile Pro Val Gly Cys Leu Asn Gly Gly 1510 1515 1520 1525 tgcggc gtg tgc aag gtg cgc gtg ctg cgc ggt gcg gtg cgc aag ctc 5064 Cys GlyVal Cys Lys Val Arg Val Leu Arg Gly Ala Val Arg Lys Leu 1530 1535 1540ggg ccg atc agc cgt gcc cat gtg agc gcg gaa gaa gag aac gac ggc 5112 GlyPro Ile Ser Arg Ala His Val Ser Ala Glu Glu Glu Asn Asp Gly 1545 15501555 tac gcg ctt gcg tgc cgc gtc gtg ccg gac ggc gac gtc gaa ctc gaa5160 Tyr Ala Leu Ala Cys Arg Val Val Pro Asp Gly Asp Val Glu Leu Glu1560 1565 1570 gtg gcc ggc cgg ctc agg aag ccg ttc ttc tgc ggc atg gcatgt gcc 5208 Val Ala Gly Arg Leu Arg Lys Pro Phe Phe Cys Gly Met Ala CysAla 1575 1580 1585 ggc acg gcg gcg atc aac aag taaccaggag gagactcaccatgggtgtga 5259 Gly Thr Ala Ala Ile Asn Lys 1590 1595 tgcgtattggtcatgtcagt ctgaaggtga tggacatgga agcggcgctg cgtcattacg 5319 tacgcgtgctcggcatgcag gaaacgatgc gcgacgcggc gggcaacgtc tacctgaaat 5379 gctgggacgaatgggacaag tattcgctga tcctgtcgcc gtccgatcag gcggggctca 5439 agcatgccgcctacaaggtc gagcacgacg ccgatctgga tgcgctgcag cagcgcatcg 5499 aagcgtacgggatcgcgacc gagatgctgc ccgaaggcgc gctgccggcg gtcggccgcc 5559 aactgcggttcctgctgccg agcggccatg aactgcggct gttcgcgaag aaggcgctgg 5619 tgggcaccgcggtcggctcg ctgaaccccg atccgtggcc cgacgacatt ccgggctcgg 5679 ccgtgcactggctcgaccac tgcctgctga tgtgcgaact gaacccggag gccggcgtga 5739 accgcgtcgaggagaacacg cgcttcatgg ccgagtgtct cgacttccat ctggccgagc 5799 aggtgatggtcggcccgggc aacacgatc 5828 2 70 PRT Burkholderia cepacia TomK polypeptide2 Met Asn Gln His Pro Thr Asp Leu Ser Pro Phe Asp Pro Gly Arg Lys 1 5 1015 Cys Val Arg Val Thr Gly Thr Asn Ala Arg Gly Phe Val Glu Phe Glu 20 2530 Leu Ser Ile Gly Gly Ala Pro Glu Leu Cys Val Glu Leu Thr Leu Ser 35 4045 Pro Ala Ala Phe Asp Ala Phe Cys Arg Glu Gln Gln Val Thr Arg Leu 50 5560 Asp Val Glu Ala Asn Pro 65 70 3 331 PRT Burkholderia cepacia TomLpolypeptide 3 Met Thr Ile Glu Leu Lys Thr Val Asp Ile Lys Pro Leu ArgHis Thr 1 5 10 15 Phe Ala His Val Ala Gln Asn Ile Gly Gly Asp Lys ThrAla Thr Arg 20 25 30 Tyr Gln Glu Gly Met Met Gly Ala Gln Pro Gln Glu AsnPhe His Tyr 35 40 45 Arg Pro Thr Trp Asp Pro Asp Tyr Glu Ile Phe Asp ProSer Arg Ser 50 55 60 Ala Ile Arg Met Ala Asn Trp Tyr Ala Leu Lys Asp ProArg Gln Phe 65 70 75 80 Tyr Tyr Ala Ser Trp Ala Thr Thr Arg Ala Arg GlnGln Asp Ala Met 85 90 95 Glu Ser Asn Phe Glu Phe Val Glu Ser Arg Arg MetIle Gly Leu Met 100 105 110 Arg Asp Asp Val Ala Ala Arg Ala Leu Asp ValLeu Val Pro Leu Arg 115 120 125 His Ala Ala Trp Gly Ala Asn Met Asn AsnAla Gln Ile Cys Ala Leu 130 135 140 Gly Tyr Gly Thr Val Phe Thr Ala ProAla Met Phe His Ala Met Asp 145 150 155 160 Asn Leu Gly Val Ala Gln TyrLeu Thr Arg Leu Ala Leu Ala Met Ala 165 170 175 Glu Pro Asp Val Leu GluAla Ala Lys Ala Thr Trp Thr Arg Asp Ala 180 185 190 Ala Trp Gln Pro LeuArg Arg Tyr Val Glu Asp Thr Leu Val Val Ala 195 200 205 Asp Pro Val GluLeu Phe Ile Ala Gln Asn Leu Ala Leu Asp Gly Leu 210 215 220 Leu Tyr ProLeu Val Tyr Asp Arg Phe Val Asp Glu Arg Ile Ala Leu 225 230 235 240 GluGly Gly Ser Ala Val Ala Met Leu Thr Ala Phe Met Pro Glu Trp 245 250 255His Thr Glu Ser Asn Arg Trp Ile Asp Ala Val Val Lys Thr Met Ala 260 265270 Ala Glu Ser Asp Asp Asn Arg Ala Leu Leu Ala Arg Trp Thr Arg Asp 275280 285 Trp Ser Ala Arg Ala Glu Ala Ala Leu Ala Pro Val Ala Ala Arg Ala290 295 300 Leu Gln Asp Ala Gly Arg Ala Ala Leu Asp Glu Val Arg Glu GlnPhe 305 310 315 320 His Ala Arg Ala Ala Arg Leu Gly Ile Ala Leu 325 3304 89 PRT Burkholderia cepacia TomM polypeptide 4 Met Ser Asn Val Phe IleAla Phe Gln Ala Asn Glu Asp Ser Arg Pro 1 5 10 15 Ile Val Asp Ala IleVal Ala Asp Asn Pro Arg Ala Val Val Val Glu 20 25 30 Ser Pro Gly Met ValLys Ile Asp Ala Pro Asp Arg Leu Thr Ile Arg 35 40 45 Arg Glu Thr Ile GluGlu Leu Thr Gly Thr Arg Phe Asp Leu Gln Gln 50 55 60 Leu Gln Val Asn LeuIle Thr Leu Ser Gly His Ile Asp Glu Asp Asp 65 70 75 80 Asp Glu Phe ThrLeu Ser Trp Ser His 85 5 516 PRT Burkholderia cepacia TomN polypeptide 5Met Asp Thr Pro Thr Leu Lys Lys Lys Leu Gly Leu Lys Asp Arg Tyr 1 5 1015 Ala Ala Met Thr Arg Gly Leu Gly Trp Glu Thr Thr Tyr Gln Pro Met 20 2530 Asp Lys Val Phe Pro Tyr Asp Arg Tyr Glu Gly Ile Lys Ile His Asp 35 4045 Trp Asp Lys Trp Val Asp Pro Phe Arg Leu Thr Met Asp Ala Tyr Trp 50 5560 Lys Tyr Gln Gly Glu Lys Glu Lys Lys Leu Tyr Ala Val Ile Asp Ala 65 7075 80 Phe Thr Gln Asn Asn Ala Phe Leu Gly Val Ser Asp Ala Arg Tyr Ile 8590 95 Asn Ala Leu Lys Leu Phe Leu Gln Gly Val Thr Pro Leu Glu Tyr Leu100 105 110 Ala His Arg Gly Phe Ala His Val Gly Arg His Phe Thr Gly GluGly 115 120 125 Ala Arg Ile Ala Cys Gln Met Gln Ser Ile Asp Glu Leu ArgHis Tyr 130 135 140 Gln Thr Glu Thr His Ala Met Ser Thr Tyr Asn Lys PhePhe Asn Gly 145 150 155 160 Phe His His Ser Asn Gln Trp Phe Asp Arg ValTrp Tyr Leu Ser Val 165 170 175 Pro Lys Ser Phe Phe Glu Asp Ala Tyr SerSer Gly Pro Phe Glu Phe 180 185 190 Leu Thr Ala Val Ser Phe Ser Phe GluTyr Val Leu Thr Asn Leu Leu 195 200 205 Phe Val Pro Phe Met Ser Gly AlaAla Tyr Asn Gly Asp Met Ser Thr 210 215 220 Val Thr Phe Gly Phe Ser AlaGln Ser Asp Glu Ser Arg His Met Thr 225 230 235 240 Leu Gly Ile Glu CysIle Lys Phe Leu Leu Glu Gln Asp Pro Asp Asn 245 250 255 Val Pro Ile ValGln Arg Trp Ile Asp Lys Trp Phe Trp Arg Gly Tyr 260 265 270 Arg Leu LeuThr Leu Val Ala Met Met Met Asp Tyr Met Gln Pro Lys 275 280 285 Arg ValMet Ser Trp Arg Glu Ser Trp Glu Met Tyr Ala Glu Gln Asn 290 295 300 GlyGly Ala Leu Phe Lys Asp Leu Ala Arg Tyr Gly Ile Arg Glu Pro 305 310 315320 Lys Gly Trp Gln Asp Ala Cys Glu Gly Lys Asp His Ile Ser His Gln 325330 335 Ala Trp Ser Thr Phe Tyr Gly Phe Asn Ala Ala Ser Ala Phe His Thr340 345 350 Trp Val Pro Thr Glu Asp Glu Met Gly Trp Leu Ser Ala Lys TyrPro 355 360 365 Asp Ser Phe Asp Arg Tyr Tyr Arg Pro Arg Phe Asp His TrpGly Glu 370 375 380 Gln Ala Arg Ala Gly Asn Arg Phe Tyr Met Lys Thr LeuPro Met Leu 385 390 395 400 Cys Gln Thr Cys Gln Ile Pro Met Leu Phe ThrGlu Pro Gly Asn Pro 405 410 415 Thr Lys Ile Gly Ala Arg Glu Ser Asn TyrLeu Gly Asn Lys Phe His 420 425 430 Phe Cys Ser Asp His Cys Lys Asp IlePhe Asp His Glu Pro Gln Lys 435 440 445 Tyr Val Gln Ala Trp Leu Pro ValHis Gln Ile His Gln Gly Asn Cys 450 455 460 Phe Pro Pro Asp Ala Asp ProGly Ala Glu Gly Phe Asp Pro Leu Ala 465 470 475 480 Ala Val Leu Asp TyrTyr Ala Val Thr Met Gly Arg Asp Asn Leu Asp 485 490 495 Phe Asp Gly SerGlu Asp Gln Lys Asn Phe Ala Ala Trp Arg Gly Gln 500 505 510 Ala Thr ArgAsn 515 6 118 PRT Burkholderia cepacia TomO polypeptide 6 Met Ala ValIle Ala Leu Lys Pro Tyr Asp Phe Pro Val Lys Asp Ala 1 5 10 15 Val GluLys Phe Pro Ala Pro Leu Leu Tyr Val Cys Trp Glu Asn His 20 25 30 Leu MetPhe Pro Ala Pro Phe Cys Leu Pro Leu Pro Pro Asp Met Pro 35 40 45 Phe GlyAla Leu Ala Gly Asp Val Leu Pro Pro Val Tyr Gly Tyr His 50 55 60 Pro AspPhe Ala Lys Ile Asp Trp Asp Arg Val Glu Trp Phe Arg Ser 65 70 75 80 GlyGlu Pro Trp Ala Pro Asp Pro Ala Lys Ser Leu Ala Gly Asn Gly 85 90 95 LeuGly His Lys Asp Leu Ile Ser Phe Arg Thr Pro Gly Leu Asp Gly 100 105 110Leu Gly Gly Ala Ser Phe 115 7 354 PRT Burkholderia cepacia TomPpolypeptide 7 Met Ser His Gln Leu Thr Ile Glu Pro Leu Gly Val Thr IleGlu Val 1 5 10 15 Glu Glu Gly Gln Thr Met Leu Asp Ala Ala Leu Arg GlnGly Ile Tyr 20 25 30 Ile Pro His Ala Cys Cys His Gly Leu Cys Gly Thr CysLys Val Ala 35 40 45 Val Leu Asp Gly Glu Thr Asp Pro Gly Asp Ala Asn ProPhe Ala Leu 50 55 60 Met Asp Phe Glu Arg Glu Glu Gly Lys Ala Leu Ala CysCys Ala Thr 65 70 75 80 Leu Gln Ala Asp Thr Val Ile Glu Ala Asp Val AspGlu Glu Pro Asp 85 90 95 Ala Glu Ile Ile Pro Val Arg Asp Phe Ala Ala AspVal Thr Arg Ile 100 105 110 Glu Gln Leu Thr Pro Thr Ile Lys Ser Ile ArgLeu Lys Leu Ser Gln 115 120 125 Pro Ile Arg Phe Gln Ala Gly Gln Tyr ValGln Leu Glu Ile Pro Gly 130 135 140 Leu Gly Gln Ser Arg Ala Phe Ser IleAla Asn Ala Pro Ala Asp Val 145 150 155 160 Ala Ala Thr Gly Glu Ile GluLeu Asn Val Arg Gln Val Pro Gly Gly 165 170 175 Leu Gly Thr Gly Tyr LeuHis Glu Gln Leu Ala Thr Gly Glu Arg Val 180 185 190 Arg Leu Ser Gly ProTyr Gly Arg Phe Phe Val Arg Arg Ser Ala Ala 195 200 205 Arg Pro Met IlePhe Met Ala Gly Gly Ser Gly Leu Ser Ser Pro Arg 210 215 220 Ser Met IleAla Asp Leu Leu Ala Ser Gly Val Thr Ala Pro Ile Thr 225 230 235 240 LeuVal Tyr Gly Gln Arg Ser Ala Gln Glu Leu Tyr Tyr His Asp Glu 245 250 255Phe Arg Ala Leu Ala Glu Arg His Pro Asn Phe Thr Tyr Val Pro Ala 260 265270 Leu Ser Glu Gly Ala Pro His Ala Gly Gly Asp Val Ala Gln Gly Phe 275280 285 Val His Asp Val Ala Lys Ala His Phe Gly Gly Asp Phe Ser Gly His290 295 300 Gln Ala Tyr Leu Cys Gly Pro Pro Ala Met Ile Asp Ala Cys IleThr 305 310 315 320 Thr Leu Met Gln Gly Arg Leu Phe Glu Arg Asp Ile TyrHis Glu Lys 325 330 335 Phe Ile Ser Ala Ala Asp Ala Gln Gln Thr Arg SerPro Leu Phe Arg 340 345 350 Arg Val 8 118 PRT Burkholderia cepacia TomQpolypeptide 8 Met Asp Ala Gly Arg Val Cys Gly Thr Val Thr Ile Ala GlnThr Asp 1 5 10 15 Glu Arg Tyr Ala Cys Val Ser Gly Glu Ser Leu Leu AlaGly Met Ala 20 25 30 Lys Leu Gly Arg Arg Gly Ile Pro Val Gly Cys Leu AsnGly Gly Cys 35 40 45 Gly Val Cys Lys Val Arg Val Leu Arg Gly Ala Val ArgLys Leu Gly 50 55 60 Pro Ile Ser Arg Ala His Val Ser Ala Glu Glu Glu AsnAsp Gly Tyr 65 70 75 80 Ala Leu Ala Cys Arg Val Val Pro Asp Gly Asp ValGlu Leu Glu Val 85 90 95 Ala Gly Arg Leu Arg Lys Pro Phe Phe Cys Gly MetAla Cys Ala Gly 100 105 110 Thr Ala Ala Ile Asn Lys 115 9 36 DNAArtificial Sequence Description of Artificial Sequence Designed PCRprimer 9 agtccgccat ggaggcgaca ccgatcatga atcagc 36 10 34 DNA ArtificialSequence Description of Artificial Sequence Designed PCR primer 10caccgaccat ggatcagcac cccaccgatc tttc 34 11 34 DNA Artificial SequenceDescription of Artificial Sequence Designed PCR primer 11 tgccgccttccatgggttct gccgcgaaca gcag 34 12 39 DNA Artificial Sequence Descriptionof Artificial Sequence Designed PCR primer 12 agcaagccat ggccatcgagctgaagacag tcgacatca 39 13 35 DNA Artificial Sequence Description ofArtificial Sequence Designed PCR primer 13 ccgaccatca cctgctcggccagatggaag tcgag 35

What is claimed is:
 1. A DNA fragment of about 5.8 Kb containing atoluene monooxygenase gene, having 1 BamHI restriction site, 2 EcoRIrestriction sites, 1 HpaI restriction site, 1 KpnI restriction site, 1NcoI restriction site, 1 NspV restriction site, 1 SacI restriction site,2 SmaI restriction sites, 3 SphI restriction sites, 2 XhoI restrictionsites, no ClaI restriction site, no DraI restriction site, no EcoRVrestriction site, no HindIII restriction site, no NdeI restriction site,no NheI restriction site, no PvuII restriction site, no ScaI restrictionsite, no Sse8387I restriction site, no StuI restriction site, and noXbaI restriction site, and having a restriction map of:


2. The DNA fragment according to claim 1, wherein the DNA fragment has anucleotide sequence of SEQ ID NO:1 in the Sequence Listing.
 3. A DNAfragment having a nucleotide sequence of SEQ ID NO:1 with deletion,substitution, and/or addition of one or more nucleotides encoding aprotein having a toluene monooxygenase activity.
 4. A recombinant DNAcomprising a vector enabling maintenance or replication in a host and aDNA fragment according to any one of claims 1 to
 3. 5. The recombinantDNA fragment according to claim 4, wherein the vector can be maintainedor replicate in a bacterium.
 6. A DNA fragment containing a regionencoding a toluene monooxygenase, the region comprising a first sequenceencoding a polypeptide TomL having an amino acid sequence of SEQ IDNO:3, a second sequence encoding a polypeptide TomM having an amino acidsequence of SEQ ID NO:4, a third sequence encoding a polypeptide TomNhaving an amino acid sequence of SEQ ID NO:5, a fourth sequence encodinga polypeptide TomO having an amino acid sequence of SEQ ID NO:6, and afifth sequence encoding a polypeptide TomP having an amino acid sequenceof SEQ ID NO:7 of the Sequence Listing, and the first to fifth sequencesare aligned so that expressed TomL-TomP polypeptides can form an activemonooxygenase protein.
 7. The DNA fragment according to claim 6, whereinno spacer sequence is present between the first to fifth sequences or atleast one spacer sequence is present between the first to fifthsequences.
 8. The DNA fragment according to claim 6 or 7, furthercomprising a sequence encoding a polypeptide TomQ having an amino acidsequence of SEQ ID NO:8 in the Sequence Listing.
 9. A DNA fragmentcontaining a region encoding a toluene monooxygenase, wherein the regioncomprises a first sequence encoding a polypeptide TomL having an aminoacid sequence of SEQ ID NO:3, a second sequence encoding a polypeptideTomM having an amino acid sequence of SEQ ID NO:4, a third sequenceencoding a polypeptide TomN having an amino acid sequence of SEQ IDNO:5, a fourth sequence encoding a polypeptide TomO having an amino acidsequence of SEQ ID NO:6, and a fifth sequence encoding a polypeptideTomP having an amino acid sequence of SEQ ID NO:7, and the first tofifth sequences are aligned so that expressed TomL-TomP polypeptides canform an active monooxygenase protein; wherein in at least one of thefirst to fifth sequences of the DNA fragment, deletion, substitution,and/or addition of one or more nucleotides are present in the provisothat the toluene monooxygenase protein is active.
 10. A DNA fragmentcomprising a region encoding a polypeptide TomK the polypeptide TomKhaving an amino acid sequence of SEQ ID NO:2, and a property to enhancethe toluene monooxygenase activity of a protein comprised of at leastTomL to TomP; or a region encoding a variant TomK in which the aminoacid sequence of SEQ ID NO:2 is altered with the proviso that theproperty to enhance the toluene monooxygenase activity is not impaired.11. A recombinant DNA comprising a vector, a promoter, and the DNAfragment according to any one of claims 6 to 9, and the vector and thepromoter are functionally ligated to the DNA fragment to enableexpression of the toluene monooxygenase encoded by the DNA fragment. 12.The recombinant DNA according to claim 11 wherein the promoter and thevector can function in a bacterium.
 13. A recombinant DNA comprising avector; a first promoter and the DNA fragment encoding polypeptide TomKaccording to claim 10 wherein the DNA fragment for TomK polypeptide isfunctionally linked to the first promoter to be expressed by the firstpromoter; a second promoter and the DNA fragment according to any one ofclaims 6 to 9 wherein the DNA fragment is functionally linked to thesecond promoter to be expressed by the second promoter.
 14. Therecombinant DNA according to claim 13, wherein the first and secondpromoters and the vector can function in a bacterium.
 15. A transformantobtained by introducing a recombinant DNA into a host microorganism, therecombinant DNA comprising a vector enabling maintenance or replicationin a host and a DNA fragment of about 5.8 Kb containing a toluenemonooxygenase gene having 1 BamHI restriction site, 2 EcoRI restrictionsites, 1 HpaI restriction site, 1 KpnI restriction site, 1 NcoIrestriction site, 1 NspV restriction site, 1 SacI restriction site, 2SmaI restriction sites, 3 SphI restriction sites, 2 XhoI restrictionsites, no ClaI restriction site, no DraI restriction site, no EcoRVrestriction site, no HindIII restriction site, no NdeI restriction site,no NheI restriction site, no PvuII restriction site, no ScaI restrictionsite, no Sse8387I restriction site, no StuI restriction site, and noXbaI restriction site, and having a restriction map of:


16. The transformant according to claim 15, wherein the hostmicroorganism is a bacterium.
 17. A transformant obtained by introducinga recombinant DNA into a host microorganism, where the recombinant DNAcomprises a vector enabling maintenance or replication in a host, and aDNA fragment ligated thereto having a nucleotide sequence of SEQ ID NO:1of the Sequence Listing with deletion, substitution and/or addition ofone or more nucleotides, still encoding an active toluene monooxygenase.18. The transformant according to claim 17, wherein the hostmicroorganism is a bacterium.
 19. A transformant obtained by introducinga recombinant DNA comprising a vector, a promoter and a DNA fragmentinto a host microorganism where the DNA fragment contains a regionencoding a toluene monooxygenase, the region comprising a first sequenceencoding a polypeptide TomL having an amino acid sequence of SEQ IDNO:3, a second sequence encoding a polypeptide TomM having an amino acidsequence of SEQ ID NO:4, a third sequence encoding a polypeptide TomNhaving an amino acid sequence of SEQ ID NO:5, a fourth sequence encodinga polypeptide TomO having an amino acid sequence of SEQ ID NO:6, and afifth sequence encoding a polypeptide TomP having an amino acid sequenceof SEQ ID NO:7, and the first to fifth sequences are aligned so thatexpressed TomL-TomP polypeptides can form an active monooxygenaseprotein; wherein the promoter and the DNA fragment are functionallylinked enabling expression of the toluene monooxygenase protein encodedby the DNA fragment.
 20. The transformant according to claim 19, whereinsaid host microorganism is a bacterium.
 21. A method for producing atoluene monooxygenase, comprising a step of making the transformantaccording to any one of claims 15, 17 and 19 produce a toluenemonooxygenase that is a gene product of the recombinant DNA introducedinto the transformant.
 22. A method for degrading at least one of achlorinated aliphatic hydrocarbon compound and an aromatic compound in amedium comprising a step of degrading at least one of a chlorinatedaliphatic hydrocarbon compound and an aromatic compound by using thetransformant according to any one of claims 15, 17 and
 19. 23. Thedegradation method according to claim 22, wherein the medium is anaqueous medium.
 24. The degradation method according to claim 22,wherein the medium is soil.
 25. The degradation method according toclaim 22, wherein the medium is air.
 26. The degradation methodaccording to claim 22, wherein the chlorinated aliphatic hydrocarboncompound is either trichloroethylene (TCE) or dichloroethylene (DCE).27. The degradation method according to claim 22, wherein the aromaticcompound is at least one of toluene, benzene, phenol, and cresol.
 28. Amethod for cleaning a medium polluted with at least one of a chlorinatedaliphatic hydrocarbon compound and aromatic compound comprising a stepof degrading at least one of a chlorinated aliphatic hydrocarboncompound and an aromatic compound using the transformant according toany one of claims 15, 17 and
 19. 29. The cleaning method according toclaim 28 wherein the medium is an aqueous medium.
 30. The cleaningmethod according to claim 28 wherein the medium is soil.
 31. Thecleaning method according to claim 28 wherein the medium is air.
 32. Thecleaning method according to claim 28 wherein the chlorinated aliphatichydrocarbon compound is either trichloroethylene (TCE) ordichloroethylene (DCE).
 33. The cleaning method according to claim 28wherein the aromatic compound is at least one of toluene, benzene,phenol, and cresol.
 34. A method for remedying an environment pollutedwith a pollutant being at least either of a chlorinated aliphatichydrocarbon compound or an aromatic compound, comprising a step ofdegrading the pollutant by using the transformant according to any oneof claims 15, 17 and
 19. 35. The remediation method according to claim34 wherein the environment is made of an aqueous medium.
 36. Theremediation method according to claim 35 wherein the polluted aqueousmedium is brought into contact with a carrier holding the transformant.37. The remediation method according to claim 36 wherein the contact iscarried out by placing the carrier holding the transformant in acontainer, introducing the polluted aqueous medium from one side of thecontainer, and discharging the remedied aqueous medium from anotherside.
 38. The remediation method according to claim 34, wherein theenvironment is made of soil.
 39. The remediation method according toclaim 38 being carried out by introducing an aqueous medium containingthe transformant into the polluted soil and supplying nutrients and/oroxygen for proliferation of the transformant in the polluted soil. 40.The remediation method according to claim 39 wherein the transformant isintroduced in the soil with applying pressure through an injection wellprovided in the polluted soil.
 41. The remediation method according toclaim 38 wherein the polluted soil is introduced in a liquid phasecontaining the transformant.
 42. The remediation method according toclaim 38 wherein the polluted soil is brought into contact with acarrier holding the transformant.
 43. The remediation method accordingto claim 34 wherein the environment is made of air.
 44. The remediationmethod according to claim 43 wherein the polluted air is introduced intoa liquid phase containing the transformant.
 45. The remediation methodaccording to claim 43 wherein the polluted air is brought into contactwith a carrier holding the transformant.
 46. The remediation methodaccording to claim 45 wherein contact is carried out by placing thecarrier holding the transformant in a container, introducing pollutedair from one side of the container, and discharging cleaned air fromanother side.
 47. The remediation method according to claim 34 whereinthe chlorinated aliphatic hydrocarbon compound is eithertrichloroethylene (TCE) or dichloroethylene (DCE).
 48. The remediationmethod according to claim 34 wherein the aromatic compound is at leastone of toluene, benzene, phenol, and cresol.
 49. A component polypeptidehaving any one of amino acid sequences of SEQ ID NOs:2 to 8 in thesequence listing, capable of being a component of a toluenemonooxygenase.
 50. A toluene monooxygenase comprising at least componentpolypeptides TomL to TomP having amino acid sequences of SEQ ID NOs:3 to7 in the Sequence Listing.
 51. The toluene monooxygenase according toclaim 50 further comprising a component polypeptide TomQ having an aminoacid sequence of SEQ ID NO:8 in the Sequence Listing.
 52. The toluenemonooxygenase according to claim 50 further comprising a componentpolypeptide TomQ having an amino acid sequence of SEQ ID NO:8 in theSequence Listing.
 53. The toluene monooxygenase according to claim 52further comprising a component polypeptide TomQ having an amino acidsequence of SEQ ID NO:8 in the Sequence Listing.
 54. A variant toluenemonooxygenase comprising at least component polypeptides TomL-TomP ofamino acid sequences of SEQ ID Nos.:3 to 7 wherein one or more aminoacids have been deleted from, substituted to, and/or added to thepolypeptides with the proviso that the enzyme activity is not impaired.55. A recombinant DNA comprising a vector, a promoter, a first DNAfragment being the DNA fragment of any one of claims 6 to 9, and asecond DNA fragment being the tomK DNA fragment of claim 10, wherein thefirst DNA fragment is functionally connected to the promoter to expressan active toluene monooxygenase, and the second DNA fragment isfunctionally connected to the promoter to express a property to enhancethe toluene monooxygenase activity.